Methods and Compositions for the Repair and/or Regeneration of Damaged Myocardium

ABSTRACT

Methods, compositions, and kits for repairing damaged myocardium and/or myocardial cells including the administration of stem cells, such as adult stem cells, optionally with cytokines are disclosed and claimed.

RELATED APPLICATIONS/PATENT & INCORPORATION BY REFERENCE

This application claims priority from Provisional U.S. PatentApplication Ser. Nos. 60/221,902 filed Jul. 31, 2000, 60/258,564 filedDec. 29, 2000 and 60/258,805 filed Jan. 2, 2001, and 60/295,807,60/295,806, 60/295, 805, 60/295,804, and 60/295,803 filed Jun. 5, 2001.

Each of the applications and patents cited in this text, including eachof the foregoing cited applications, as well as each document orreference cited in each of the applications and patents (includingduring the prosecution of each issued patent; “application citeddocuments”), and each of the PCT and foreign applications or patentscorresponding to and/or claiming priority from any of these applicationsand patents, and each of the documents cited or referenced in each ofthe application cited documents, are hereby expressly incorporatedherein by reference. More generally, various documents or references arecited in this text, either in a Reference List before the claims or inthe text itself; and, each of the documents or references (“herein citeddocuments”) and all of the documents cited in this text (also “hereincited documents”), as well as each document or reference cited in eachof the herein cited documents (including any manufacturer'sspecifications, instructions, etc. for products mentioned herein and inany document incorporated herein by reference), is hereby expresslyincorporated herein by reference. There is no admission that any of thevarious documents cited in this text are prior art as to the presentinvention. Any document having as an author or inventor person orpersons named as an inventor herein is a document that is not by anotheras to the inventive entity herein. Also, teachings of herein citeddocuments and documents cited in herein cited documents and moregenerally in all documents incorporated herein by reference can beemployed in the practice and utilities of the present invention.

FIELD OF THE INVENTION

The present invention relates generally to the field of cardiology, andmore particularly relates to methods and cellular compositions fortreatment of a patient suffering from a cardiovascular disease,including, but not limited to, atherosclerosis, ischemia, hypertension,restenosis, angina pectoris, rheumatic heart disease, congenitalcardiovascular defects and arterial inflammation and other disease ofthe arteries, arterioles and capillaries.

Moreover, the present invention relates to any one or more of:

Methods and/or pharmaceutical composition comprising a therapeuticallyeffective amount of somatic stem cells alone or in combination with acytokine such as a cytokine selected from the group consisting of stemcell factor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), stromalcell-derived factor-1, steel factor, vascular endothelial growth factor,macrophage colony stimulating factor, granulocyte-macrophage stimulatingfactor or Interleukin-3 or any cytokine capable of the stimulatingand/or mobilizing stem cells. Cytokines may be administered alone or incombination of with any other cytokine capable of: the stimulationand/or mobilization of stem cells; the maintenance of early and latehematopoiesis (see below); the activation of monocytes (see below),macrophage/monocyte proliferation; differentiation, motility andsurvival (see below) and a pharmaceutically acceptable carrier, diluentor excipient (including combinations thereof). The stem cells areadvantageously adult stem cells, such as hematopoietic or cardiac stemcells or a combination thereof or a combination of cardiac stem cellsand any other type of stem cells.

The implanting, depositing, administering or causing of implanting ordepositing or administering of stem cells, such as adult stem cells, forinstance hematopoietic or cardiac stem cells or a combination thereof orany combination of cardiac stem cells (e.g., adult cardiac stem cells)and stem cells of another type of (e.g., adult stem cells of anothertype), alone or with a cytokine such as a cytokine selected from thegroup consisting of stem cell factor (SCF), granulocyte-colonystimulating factor (G-CSF), granulocyte-macrophage colony stimulatingfactor (GM-CSF), stromal cell-derived factor-1, steel factor, vascularendothelial growth factor, macrophage colony stimulating factor,granulocyte-macrophage stimulating factor or Interleukin-3 or anycytokine capable of the stimulating and/or mobilizing stem cells(wherein “with a cytokine . . . ” can include sequential implanting,depositing administering or causing of implanting or depositing oradministering of the stem cells and the cytokine or the co-implantingco-depositing or co-administering or causing of co-implanting orco-depositing or co-administering or the simultaneous implanting,depositing administering or causing of implanting or depositing oradministering of the stem cells and the cytokine), in circulatory tissueor muscle tissue or circulatory muscle tissue, e.g., cardiac tissue,such as the heart or blood vessels—e.g., veins, arteries, that go to orcome from the heart such as veins and arteries directly connected orattached or flowing into the heart, for instance the aorta. Thisimplanting, depositing, or administering or causing of implanting,depositing or administering can be in conjunction with grafts. Suchimplanting, depositing or administering or causing of implanting,depositing or administering is advantageously employed in the treatmentor therapy or prevention of cardiac conditions, such as to treat areasof weakness or scarring in the heart or prevent the occurrence orfurther occurrence of such areas or to treat conditions which cause orirritate such areas, for instance myocardial infarction or ischemia orother e.g., genetic, conditions that impart weakness or scarring to theheart (see also cardiac conditions mentioned infra).

The use of such stem cells alone or in combination with saidcytokine(s), in the formulation of medicaments for such treatment,therapy or prevention.

Medicaments for use in such treatment, therapy or prevention comprisingthe stem cells and optionally the cytokine(s).

Kits comprising the stem cells and optionally the cytokine(s) forformulations for use in such treatment, therapy or prevention.

Compositions comprising such stem cells and optionally at least onecytokine and kits for preparing such compositions.

Methods of making the kits and compositions described herein.

Methods of implanting or depositing stem cells or causing the implantingor depositing of stem cells.

BACKGROUND OF THE INVENTION

Cardiovascular disease is a major health risk throughout theindustrialized world. Atherosclerosis, the most prevalent ofcardiovascular diseases, is the principal cause of heart attack, stroke,and gangrene of the extremities, and thereby the principal cause ofdeath in the United States. Atherosclerosis is a complex diseaseinvolving many cell types and molecular factors (for a detailed review,see Ross, 1993, Nature 362: 801-809).

Ischemia is a condition characterized by a lack of oxygen supply intissues of organs due to inadequate perfusion. Such inadequate perfusioncan have number of natural causes, including atherosclerotic orrestenotic lesions, anemia, or stroke, to name a few. Many medicalinterventions, such as the interruption of the flow of blood duringbypass surgery, for example, also lead to ischemia. In addition tosometimes being caused by diseased cardiovascular tissue, ischemia maysometimes affect cardiovascular tissue, such as in ischemic heartdisease. Ischemia may occur in any organ, however, that is suffering alack of oxygen supply.

The most common cause of ischemia in the heart is myocardial infarction(MI), commonly known as a heart attack, is one of the most well-knowntypes of cardiovascular disease. 1998 estimates show 7.3 million peoplein the United States suffer from MI, with over one million experiencingan MI in a given year (American Heart Association, 2000). Of theseindividuals, 25% of men, and 38% of females will die within a year oftheir first recognized MI (American Heart Association, 2000). MI iscaused by a sudden and sustained lack of blood flow to an area of theheart, commonly caused by narrowing of a coronary artery. Withoutadequate blood supply, the tissue becomes ischemic, leading to the deathof myocytes and vascular structures. This area of necrotic tissue isreferred to as the infarct site, and will eventually become scar tissue.

Current treatments for MI focus on reperfusion therapy, which attemptsto start the flow of blood to the affected area to prevent the furtherloss of tissue. The main choices for reperfusion therapy include the useof anti-thrombolytic agents, or performing balloon angioplasty, or acoronary artery bypass graft. Anti-thrombolytic agents solubilize bloodclots that may be blocking the artery, while balloon angioplasty threadsa catheter into the artery to the site of the occlusion, where the tipof the catheter is inflated, pushing open the artery. Still moreinvasive procedures include the bypass, where surgeons remove a sectionof a vein from the patient, and use it to create a new artery in theheart, which bypasses the blockage, and continues the supply of blood tothe affected area. In 1998, there were an estimated 553,000 coronaryartery bypass graft surgeries and 539,000 percutaneous transluminalcoronary angioplastys. These procedures average $27,091 and $8,982 perpatient, respectively (American Heart Association, 2000).

These treatments may succeed in reestablishing the blood supply, howevertissue damage that occurred before the reperfusion treatment began hasbeen thought to be irreversible. For this reason, eligible MI patientsare started on reperfusion therapy as soon as possible to limit the areaof the infarct.

As such, most studies on MI have also focused on reducing infarct size.There have been a few attempts to regenerate the necrotic tissue bytransplanting cardiomyocytes or skeletal myoblasts (Leor et al., 1996;Murray, et al., 1996; Taylor, et al., 1998; Tomita et al., 1999;Menasche et al., 2000). While the cells may survive aftertransplantation, they fail to reconstitute healthy myocardium andcoronary vessels that are both functionally and structurally sound.

All of the cells in the normal adult originate as precursor cells whichreside in various sections of the body. These cells, in turn, derivefrom very immature cells, called progenitors, which are assayed by theirdevelopment into contiguous colonies of cells in 1-3 week cultures insemisolid media such as methylcellulose or agar. Progenitor cellsthemselves derive from a class of progenitor cells called stem cells.Stem cells have the capacity, upon division, for both self-renewal anddifferentiation into progenitors. Thus, dividing stem cells generateboth additional primitive stem cells and somewhat more differentiatedprogenitor cells. In addition to the well-known role of stem cells inthe development of blood cells, stem cells also give rise to cells foundin other tissues, including but not limited to the liver, brain, andheart.

Stem cells have the ability to divide indefinitely, and to specializeinto specific types of cells. Totipotent stem cells, which exist afteran egg is fertilized and begins dividing, have total potential, and areable to become any type of cell. Once the cells have reached theblastula stage, the potential of the cells has lessened, with the cellsstill able to develop into any cell within the body, however they areunable to develop into the support tissues needed for development of anembryo. The cells are considered pluripotent, as they may still developinto many types of cells. During development, these cells become morespecialized, committing to give rise to cells with a specific function.These cells, considered multipotent, are found in human adults andreferred to as adult stem cells. It is well known that stem cells arelocated in the bone marrow, and that there is a small amount ofperipheral blood stem cells that circulate throughout the blood stream(National Institutes of Health, 2000).

Due to the regenerative properties of stem cells, they have beenconsidered an untapped resource for potential engineering of tissues andorgans. It would be an advance to provide uses of stem cells withrespect to addressing cardiac conditions.

Mention is made of:

U.S. Pat. No. 6,117,675 which relates to the differentiation of retinalstem cells into retinal cells in vivo or in vitro, which can be used asa therapy to restore vision.

U.S. Pat. No. 6,001,934 involving the development of functional isletsfrom islets of Langerhans stem cells.

U.S. Pat. Nos. 5,906,934 and 6,174,333 pertaining to the use ofmesenchymal stem cells for cartilage repair, and the use of mesenchymalstem cells for regeneration of ligaments; for instance, wherein the stemcells are embedded in a gel matrix, which is contracted and thenimplanted to replace the desired soft tissue.

U.S. Pat. Nos. 6,099,832, and 6,110,459 involving grafts with celltransplantation.

PCT Application Nos. PCT/US00/08353 (WO 00/57922) and PCT/US99/17326 WO00/06701) involving intramyocardial injection of autologous bone marrowand mesenchymal stem cells which fails to teach or suggestadministering, implanting, depositing or the use of hematopoietic stemcells as in the present invention, especially as hematopoietic stemcells as in the present invention are advantageously isolated and/orpurified adult hematopoietic stem cells.

Furthermore, at least certain of these patent documents fail to teach orsuggest the present invention for additional reasons. The source of thestem cells of interest is limited to the known precursors of the type oftissue for which regeneration is required. Obtaining and purifying thesespecific cells can be extremely difficult, as there are often very fewstem cells in a given tissue. In contrast, a benefit of the presentinvention results from the ability of various lineages of stem cells tohome to the myocardium damage and differentiate into the appropriatecell types—an approach that does not require that the stem cells arerecovered directly from myocardium, and, a variety of types of stemcells may be used without compromising the functionality of theregenerated tissue. And, other of these patent documents utilize stemcells as the source of various chemical compositions, without utilizingtheir proliferative capabilities, and thereby fail to teach or suggestthe invention.

Only recent literature has started to investigate the potentials forstem cells to aid in the repair of tissues other than that of knownspecialization. This plasticity of stem cells, the ability to cross theborder of germ layers, is a concept only in its infancy (Kempermann etal, 2000, Temple, 2001). Kocher et al (2001) discusses the use of adultbone marrow to induce neovascularization after infarction as analternative therapy for left ventricle remodeling (reviewed in Rosenthaland Tsao, 2001). Other studies have focused on coaxing specific types ofstem cells to differentiate into myocardial cells, i.e. liver stem cellsas shown in Malour et al (2001). Still other work focuses on thepossibilities of bone-marrow derived stem cells (Krause, et al., 2001).

One of the oldest uses of stem cells in medicine is for the treatment ofcancer. In these treatments, bone marrow is transplanted into a patientwhose own marrow has been destroyed by radiation, allowing the stemcells in the transplanted bone marrow to produce new, healthy, whiteblood cells.

In these treatments, the stem cells are transplanted into their normalenvironment, where they continue to function as normal. Until recently,it was thought that any particular stem cell line was only capable ofproducing three or four types of cells, and as such, they were onlyutilized in treatments where the stem cell was required to become one ofthe types of cells for which their ability was already proven.Researchers are beginning to explore other options for treatments ofmyriad disorders, where the role of the stem cell is not well defined.Examples of such work will be presented in support of the presentinvention.

Organ transplantation has been widely used to replace diseased,nonfunctional tissue. More recently, cellular transplantation to augmentdeficiencies in host tissue function has emerged as a potentialtherapeutic paradigm. One example of this approach is the wellpublicized use of fetal tissue in individuals with Parkinsonism(reviewed in Tompson, 1992), where dopamine secretion from transplantedcells alleviates the deficiency in patients. In other studies,transplanted myoblasts from uneffected siblings fused with endogenousmyotubes in Duchenne's patients; importantly the grafted myotubesexpressed wild-type dystrophin (Gussoni et al., 1992).

Despite their relevance in other areas, these earlier studies do notdescribe any cellular transplantation technology that can besuccessfully applied to the heart, where the ability to replace damagedmyocardium would have obvious clinical relevance. Additionally, the useof intra-cardiac grafts to target the long-term expression of angiogenicfactors and ionotropic peptides would be of therapeutic value forindividuals with myocardial ischemia or congestive heart failure,respectively.

In light of this background there is a need for the improvement ofmyocardial regeneration technology in the heart. Desirably, suchtechnology would not only result in tissue regeneration in the heart butalso enable the delivery of useful compositions directly to the heart.The present invention addresses these needs.

It is therefore believed that heretofore the administration, implanting,depositing, causing to be deposited, implanted or administered of stemcells, alone or in combination with at least one cytokine, as well asthe use of such stem cells alone or in combination with saidcytokine(s), in the formulation of medicaments for treatment, therapy orprevention, as in this disclosure and as in the present invention, hasnot been taught, or suggested in the art and that herein methods,compositions, kits and uses are novel, nonobvious and inventive, i.e.,that the present invention has not been taught or suggested in the artand that the present invention is novel, nonobvious and inventive.

OBJECT AND SUMMARY OF THE INVENTION

It has surprisingly been found that the implantation of somatic stemcells into the myocardium surrounding an infarct following a myocardialinfarction, migrate into the damaged area, where they differentiate intomyocytes, endothelial cells and smooth muscle cells and then proliferateand form structures including myocardium, coronary arteries, arterioles,and capillaries, restoring the structural and functional integrity ofthe infarct.

It has also surprisingly been found that following a myocardialinfarction, the administration of a cytokine to the patient, stimulatesthe patient's own stem cells, causing them to enter the blood stream andhome to the infarcted area. It has also been found that once the cellshome to the infarct, they migrate into the damaged tissue, where theydifferentiate into myocytes, endothelial cells and smooth muscle cellsand then proliferate and form structures including myocardium, coronaryarteries, arterioles and capillaries, restoring structural andfunctional integrity to the infracted area.

The invention provides to methods and/or compositions for repairingand/or regenerating recently damaged myocardium and/or myocardial cellscomprising the administration of somatic stem cells, e.g., adult stemcells or cardiac stem cells or hematopoietic stem cells or a combinationthereof, such as adult cardiac or adult hematopoietic stem cells or acombination thereof or a combination of cardiac stem cells and a stemcell of another type, such as a combination of adult cardiac stem cellsand adult stem cells of another type.

The invention further provides a method and/or compositions forrepairing and/or regenerating recently damaged myocardium and/ormyocardial cells comprising the administration of at least one cytokine.

The invention still further relates to a method and/or compositions forrepairing and/or regenerating recently damaged myocardium comprising theadministration of somatic stem cells, e.g., adult stem cells or cardiacstem cells or hematopoietic stem cells or a combination thereof, such asadult cardiac or adult hematopoietic stem cells or a combination thereofor a combination of cardiac stein cells and a stem cell of another type,such as a combination of adult cardiac stem cells and adult stem cellsof another type and a cytokine.

The invention yet further provides a method for preparing any of theaforementioned or herein disclosed compositions comprising admixing thepharmaceutically acceptable carrier and the somatic stem cells and/orcytokines.

The invention also provides to a kit comprising a pharmaceuticalcomposition for use in repairing and/or regenerating recently damagedmyocardium and/or myocardial cells.

The invention provides methods involving implanting, depositing,administering or causing the implanting or depositing or administeringof stem cells, such as adult stem cells, for instance hematopoietic orcardiac stem cells or a combination thereof or any combination ofcardiac stem cells (e.g., adult cardiac stem cells) and stem cells ofanother type of (e.g., adult stem cells of another type), alone or witha cytokine such as a cytokine selected from the group consisting of stemcell factor (SCF), granulocyte-colony stimulating factor (G-CSF),granulocyte-macrophage colony stimulating factor (GM-CSF), stromalcell-derived factor-1, steel factor, vascular endothelial growth factor,macrophage colony stimulating factor, granulocyte-macrophage stimulatingfactor or Interleukin-3 or any cytokine capable of the stimulatingand/or mobilizing stem cells (wherein “with a cytokine . . . ” caninclude sequential implanting, depositing administering or causing ofimplanting or depositing or administering of the stem cells and thecytokine or the co-implanting co-depositing or co-administering orcausing of co-implanting or co-depositing or co-administering or thesimultaneous implanting, depositing administering or causing ofimplanting or depositing or administering of the stem cells and thecytokine), in circulatory tissue or muscle tissue or circulatory muscletissue, e.g., cardiac tissue, such as the heart or blood vessels—e.g.,veins, arteries, that go to or come from the heart such as veins andarteries directly connected or attached or flowing into the heart, forinstance the aorta. This implanting, depositing, or administering orcausing of implanting, depositing or administering can be in conjunctionwith grafts.

Such implanting, depositing or administering or causing of implanting,depositing or administering is advantageously employed in the treatmentor therapy or prevention of cardiac conditions, such as to treat areasof weakness or scarring in the heart or prevent the occurrence orfurther occurrence of such areas or to treat conditions which cause orirritate such areas, for instance myocardial infarction or ischemia orother e.g., genetic, conditions that impart weakness or scarring to theheart (see also cardiac conditions mentioned supra).

The invention additionally provides the use of such stem cells alone orin combination with said cytokine(s), in the formulation of medicamentsfor such treatment, therapy or prevention.

And thus, the invention also provides medicaments for use in suchtreatment, therapy or prevention comprising the stem cells andoptionally the cytokine(s).

Likewise the invention provides kits comprising the stem cells andoptionally the cytokine(s) for formulations for use in such treatment,therapy or prevention. The stem cells and the cytokine(s) can be inseparate containers in a package or in one container in a package; and,the kit can optionally include a device for administration (e.g.,syringe) and/or instructions for administration and/or admixture.

The invention also provides compositions comprising such stem cells andoptionally the cytokine(s) and kits for preparing such compositions(e.g., kits comprising the stem cells and optionally the cytokine(s);stem cells and the cytokine(s) can be in separate containers in apackage or in one container in a package; and, the kit can optionallyinclude a device for administration (e.g., syringe) and/or instructionsfor administration and/or admixture), as well as methods of making theaforementioned compositions.

The invention also provides a means of generating and/or regeneratingmyocardium ex vivo, wherein somatic stem cells and heart tissue arecultured in vitro, optionally in the presence of a cytokine. The somaticstem cells differentiate into myocytes, smooth muscle cells andendothelial cells, and proliferate in vitro, forming myocardial tissueand/or cells. These tissues and cells may assemble into cardiacstructures including arteries, arterioles, capillaries, and myocardium.The tissue and/or cells formed in vitro may then be implanted into apatient, e.g. via a graft, to restore structural and functionalintegrity.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF FIGURES

The following Detailed Description, given to describe the invention byway of example, but not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figures, incorporated herein by reference, in which:

FIG. 1 shows a log-log plot showing Lin⁻ bone marrow cells from EGFPtransgenic mice sorted by FACS based on c-kit expression (The fractionof c-kit^(POS) cells (upper gate) was 6.4%. c-kit^(NEG) cells are shownin the lower gate. c-kit^(POS) cells were 1-2 logs brighter thanc-kit^(NEG) cells)

FIG. 2A shows a photograph of a tissue section from a MI induced mouse(The photograph shows the area of myocardial infarct (MI) injected withLin⁻c-kit^(POS) cells from bone marrow (arrows), the remaining viablemyocardium (VM), and the regenerating myocardium (arrowheads).Magnification is 12×);

FIG. 2B shows a photograph of the same tissue section of FIG. 2A at ahigher magnification, centering on the area of the MI with magnificationbeing 50×;

FIGS. 2C, D show photographs of a tissue section at low and highmagnifications of the area of MI, injected with Lin⁻c-kit^(POS) cells,with the magnification of 2C being 25×, and the magnification of 2Dbeing 50×;

FIG. 2E shows a photograph of a tissue section of the area of MIinjected with Lin⁻c-kit^(NEG) cells wherein only healing is apparent andthe magnification is 50× (*Necrotic myocytes. Red=cardiac myosin;green=PI labeling of nuclei);

FIGS. 3A-C show photographs of a section of tissue from a MI inducedmouse, showing the area of MI injected with Lin⁻c-kit^(POS) cells(Visible is a section of regenerating myocardium from endocardium (EN)to epicardium (EP). All photographs are labeled to show the presence ofinfarcted tissue in the subendocardium (IT) and spared myocytes in thesubendocardium (SM). FIG. 3A is stained to show the presence of EGFP(green). Magnification is 250×. FIG. 3B is stained to show the presenceof cardiac myosin (red). Magnification is 250×. FIG. 3C is stained toshow the presence of both EGFP and myosin (red-green), as well asPI-stained nuclei (blue). Magnification is 250×);

FIG. 4A shows of grafts depicting the effects of myocardial infarctionon left ventricular end-diastolic pressure (LVEDP), developed pressure(LVDP), LV+rate of pressure rise (dP/dt), and LV−rate of pressure decay(dP/dt) (From left to right, bars indicate: sham-operated mice (SO,n=11); mice non-injected with Lin⁻c-kit^(POS) cells (MI, n=5 injectedwith Lin⁻c-kit^(NEG) cells; n=6 non-injected); mice injected withLin⁻c-kit^(POS) cells (MI+BM, n=9). Error bars are the standarddeviation. *^(,) † p<0.05 vs SO and MI);

FIG. 4B shows a drawing of a proposed scheme for Lin⁻c-kit^(POS) celldifferentiation in cardiac muscle and functional implications;

FIGS. 5A-I show photographs of a tissue sections from a MI induced mousedepicting regenerating myocardium in the area of the MI which has beeninjected with Lin⁻c-kit^(POS) cells (FIG. 5A is stained to show thepresence of EGFP (green). Magnification is 300×. FIG. 5B is stained toshow the presence of α-smooth muscle actin in arterioles (red).Magnification is 300×. FIG. 5C is stained to show the presence of bothEGFP and α-smooth muscle actin (yellow-red), as well as PI-stainednuclei (blue). Magnification is 300×. FIGS. 5D-F and G-I depict thepresence of MEF2 and Csx/Nkx2.5 in cardiac myosin positive cells. FIG.5D shows PI-stained nuclei (blue). Magnification is 300×. FIG. 5E isstained to show MEF2 and Csx/NRx2.5 labeling (green). Magnification is300×. FIG. 5F is stained to show cardiac myosin (red), as well as MEF2or Csx/NRx2.5 with PI (bright fluorescence in nuclei). Magnification is300×. FIG. 5G shows PI-stained nuclei (blue). Magnification is 300×.FIG. 5H is stained to show MEF2 and Csx/NRx2.5 labeling (green).Magnification is 300×. FIG. 5I is stained to show cardiac myosin (red),as well as MEF2 or Csx/NRx2.5 with PI (bright fluorescence in nuclei).Magnification is 300×);

FIG. 6 (FIGS. 6A-F) shows photographs of tissue sections from MI inducedmice, showing regenerating myocardium in the area of the MI injectedwith Lin⁻c-kit^(POS) cells (FIGS. 6A-C show tissue which has beenincubated in the presence of antibodies to BrdU. FIG. 6A has beenstained to show PI-labeled nuclei (blue). Magnification is 900×. FIG. 6Bhas been stained to show BrdU- and Ki67-labeled nuclei (green).Magnification is 900×. FIG. 6C has been stained to show the presence ofα-sarcomeric actin (red). Magnification is 900×. FIGS. 6D-F shows tissuethat has been incubated in the presence of antibodies to Ki67. FIG. 6Dhas been stained to show PI-labeled nuclei (blue). Magnification is500×. FIG. 6E has been stained to show BrdU- and Ki67-labeled nuclei(green). Magnification is 500×. FIG. 6F has been stained to show thepresence of α-smooth muscle actin (red). Magnification is 500×. Brightfluorescence: combination of PI with BrdU (C) or Ki67 (F));

FIG. 7 (FIGS. 7A-C) shows photographs of tissue sections from MI inducedmice, showing the area of MI injected with Lin⁻c-kit^(POS) cells(Depicted are the border zone, viable myocardium VM) and the new band(NB) of myocardium separated by an area of infarcted non-repairingtissue (arrows). FIG. 7A is stained to show the presence of EGFP(green). Magnification is 280×. FIG. 7B is stained to show the presenceof cardiac myosin (red). Magnification is 280×. FIG. 7C is stained toshow the presence of both EGFP and myosin (red-green), as well asPI-stained nuclei (blue). Magnification is 280×);

FIG. 8 (FIGS. 8A-F) shows photographs of tissue sections from MI inducedmice, showing regenerating myocardium in the area of MI injected withLin⁻c-kit^(POS) cells (FIG. 8A is stained to show the presence of EGFP(green). Magnification is 650×. FIG. 8B is stained to show the presenceof cardiac myosin (red). Magnification is 650×. FIG. 8C is stained toshow both the presence of EGFP and myosin (yellow), as well asPI-stained nuclei (blue). Magnification is 650×. FIG. 8D is stained toshow the presence of EGFP (green). Magnification is 650×. FIG. 8E isstained to show the presence of α-smooth muscle actin in arterioles(red). Magnification is 650×. FIG. 8F is stained to show the presence ofboth EGFP and α-smooth muscle actin (yellow-red) as well as PI-stainednuclei (blue). Magnification is 650×);

FIG. 9 (FIGS. 9A-C) shows photographs of tissue sections from MI inducedmice, showing the area of MI injected with Lin⁻c-kit^(POS) cells andshowing regenerating myocardium (arrowheads). (FIG. 9A is stained toshow the presence of cardiac myosin (red) Magnification is 400×. FIG. 9Bis stained to show the presence of the Y chromosome (green).Magnification is 400×. FIG. 9C is stained to show both the presence ofthe Y chromosome (light blue) and PI-labeled nuclei (dark blue). Notethe lack of Y chromosome in infarcted tissue (IT) in subendocardium andspared myocytes (SM) in subepicardium. Magnification is 400×);

FIG. 10 (FIGS. 10A-C) shows photographs of tissue sections from MIinduced mice, showing GATA-4 in cardiac myosin positive cells (FIG. 10Ashows PI-stained nuclei (blue). Magnification is 650×. FIG. 10B showsthe presence of GATA-4 labeling (green). Magnification is 650×. FIG. 10Cis stained to show cardiac myosin (red) in combination with GATA-4 andPI (bright fluorescence in nuclei). Magnification is 650×);

FIG. 11 (FIG. 11A-D)) shows photograph of tissue sections from a MIinduced mouse (FIG. 11A shows the border zone between the infarctedtissue and the surviving tissue. Magnification is 500×. FIG. 11B showsregenerating myocardium. Magnification is 800×. —FIG. 11C is stained toshow the presence of connexin 43 (yellow-green), and the contactsbetween myocytes are shown by arrows. Magnification is 800×. FIG. 11D isstained to show both α-sarcomeric actin (red) and PI-stained nuclei(blue). Magnification is 800×);

FIG. 12 (FIGS. 12A-B) shows photographs of tissue sections from a MIinduced mouse showing the area of MI that was injected withLin⁻c-kit^(POS) cells and now shows regenerating myocytes (FIG. 12A isstained to show the presence of cardiac myosin (red) and PI-labelednuclei (yellow-green). Magnification is 1,000. FIG. 12B is the same asFIG. 12A at a magnification of 700×);

FIGS. 13A-B show photographs of tissue sections from MI induced mice(FIG. 13A shows a large infarct (MI) in a cytokine-treated mouse withforming myocardium (arrowheads) (Magnification is 50×) at highermagnification (80×-adjacent panel). FIG. 13B shows a MI in a non-treatedmouse. Healing comprises the entire infarct (arrowheads) (Magnificationis 50×). Scarring is seen at higher magnification (80×-adjacent panel).Red=cardiac myosin; yellow-green=propidium iodide (PI) labeling ofnuclei; blue-magenta-collagen types I and III);

FIG. 13C shows a graph showing the mortality and myocardial regenerationin treated and untreated MI induced mice (Cytokine-treated infarctedmice, n=15; untreated infarcted mice, n=52. Log-rank test: p<0.0001);

FIG. 14 shows a graph showing quantitative measurement of infarct size(Total number of myocytes in the left ventricular free wall (LVFW) ofsham-operated (SO, n=9), infarcted non-treated (MI, n=9) andcytokine-treated (MI-C, n=11) mice at sacrifice, 27 days afterinfarction or sham operation. The percentage of myocytes lost equalsinfarct size. X±SD, *p<0.05 vs SO);

FIGS. 15A-C show graphs comparing aspects of myocardial infarction,cardiac anatomy and function (FIGS. 15 A-C depict LV dimensions atsacrifice, 27 days after surgery; sham-operated (SO, n=9), non-treatedinfarcted (MI, n=9) and cytokine-treated infarcted (MI-C, n=10));

FIG. 15D shows EF by echocardiography; (SO, n=9; MI, n=9; and MI, n=9);

FIGS. 15E-M show M-mode echocardiograms of SO (e-g), MI (h-j) and MI-C(k-m) (Newly formed contracting myocardium (arrows));

FIG. 15N shows a graph showing wall stress; SO (n=9), MI (n=8) and MI-C(n=9) (Results are mean ±SD. *^(,)**p<0.05 vs SO and MI, respectively);

FIGS. 16A-G show grafts depicting aspects of myocardial infarction,cardiac anatomy and ventricular function (FIGS. 16A-D showechocardiographic LVESD (a), LVEDD (b), PWST (c) and PWDT (d) in SO(n=9), MI (n=9) and MI-C (n=9). FIGS. 16E-G show mural thickness (e),chamber diameter (f) and longitudinal axis (g) measured anatomically atsacrifice in SO (n=9), MI (n=9) and MI-C (n=10). ***p<0.05 vs SO and MI,respectively;

FIGS. 16H-P show two dimensional (2D) images and M-mode tracings of SO(h-j), MI (k-m) and MI-C (n-p);

FIG. 17 (FIGS. 17A-D) shows graphs depicting aspects of ventricularfunction (FIG. 17A-D show LV hemodynamics in anesthetized mice atsacrifice, 27 days after infarction or sham operation; SO (n=9), MI(n=9) and MI-C (n=10). For symbols and statistics, see also FIG. 13);

FIG. 18A-E shows graphs of aspects of myocardial regeneration (FIG. 18Aclassifies the cells in the tissue as remaining viable (Re), lost (Lo)and newly formed (Fo) myocardium in LVFW at 27 days in MI and MI-C; SO,myocardium without infarct. FIG. 18B shows the amount of cellularhypertrophy in spared myocardium. FIG. 18C shows cell proliferation inthe regenerating myocardium. Myocytes (M), EC and SMC labeled by BrdUand Ki67; n=11. *^(,)**p<0.05 vs M and EC. FIGS. 18D-E depict thevolume, number (n=11) and class distribution (bucket size, 100 μm³;n=4,400) of myocytes within the formed myocardium;

FIGS. 18F-H show photographs of tissue sections from MI induced micedepicting arterioles with TER-119 labeled erythrocyte membrane (greenfluorescence); blue fluorescence=PI staining of nuclei; redfluorescence=α-smooth muscle actin in SMC (FIG. 18F is magnified at800×. FIGS. 18G-H are magnified at 1,200×);

FIG. 19 (FIGS. 19A-D) shows photographs of tissue sections from MIinduced mice that were incubated with antibodies to Ki67 (A,B) and BrdU(C,D) (FIG. 19A shows labeling of myocytes by cardiac myosin. Brightfluorescence of nuclei reflects the combination of PI and Ki67.Magnification is 800×. FIG. 19B shows labeling of SMC by α-smooth muscleactin. Bright fluorescence of nuclei reflects the combination of PI andKi67. Magnification is 1,200×.

FIG. 19C shows labeling of SMC by α-smooth muscle actin. Brightfluorescence of nuclei reflects the combination of PI and BrdU.Magnification is 1,200×. FIG. 19D shows labeling of EC in the formingmyocardium by factor VIII. Bright fluorescence of nuclei reflects thecombination of PI and BrdU. Magnification is 1,600×;

FIG. 20 (FIGS. 20A-F) shows photographs of tissue sections from MIinduced mice showing markers of differentiating cardiac cells (FIG. 20Ais stained to show labeling of myocytes by nestin (yellow)). Redfluorescence indicates cardiac myosin. Magnification is 1,200×. FIG. 20B is stained to show labeling of desmin (red). Magnification is 800×.FIG. 20C is stained to show labeling of connexin 43 (green). Redfluorescence indicates cardiac myosin. Magnification is 1,400×. FIG. 20Dshows VE-cadherin and yellow-green fluorescence reflects labeling of ECby flk-1 (arrows). Magnification is 1,800×. FIG. 20E shows redfluorescence indicating factor VIII in EC and yellow-green fluorescencereflects labeling of EC by flk-1 (arrows). Magnification is 1,200×. FIG.20F shows green fluorescence labeling of SMC cytoplasms by flk-1 andendothelial lining labeled by flk-1. Red fluorescence indicates α-smoothmuscle actin. Blue fluorescence indicates PI labeling of nuclei.Magnification is 800×; and

FIG. 21A-C show tissue sections from MI induced mice (FIG. 21A usesbright fluorescence to depict the combination of PI labeling of nucleiwith Csx/NRx2.5. Magnification is 1,400×. FIG. 21B uses brightfluorescence to depict the combination of PI labeling of nuclei withGATA-4. Magnification is 1,200×. FIG. 21C uses bright fluorescence todepict the combination of PI labeling of nuclei with MEF2. Magnificationis 1,200× (Red fluorescence shows cardiac myosin antibody staining andblue fluorescence depicts PI labeling of nuclei. The fraction of myocytenuclei labeled by Csx/NRx2.5, GATA-4 and MEF2 was 63±5% (nucleisampled=2,790; n=11), 94±9% (nuclei sampled=2,810, n=11) and 85±14%(nuclei sampled=3,090; n=11), respectively).

DETAILED DESCRIPTION

The present invention provides methods and/or pharmaceutical compositioncomprising a therapeutically effective amount of somatic stem cellsalone or in combination with a cytokine selected from the groupconsisting of stem cell factor (SCF), granulocyte-colony stimulatingfactor (G-CSF), granulocyte-macrophage colony stimulating factor(GM-CSF), stromal cell-derived factor-1, steel factor, vascularendothelial growth factor, macrophage colony stimulating factor,granulocyte-macrophage stimulating factor or Interleukin-3 or anycytokine capable of the stimulating and/or mobilizing stem cells.Cytokines may be administered alone or in combination or with any othercytokine capable of: the stimulation and/or mobilization of stem cells;the maintenance of early and late hematopoiesis (see below); theactivation of monocytes (see below), macrophage/monocyte proliferation;differentiation, motility and survival (see below) and apharmaceutically acceptable carrier, diluent or excipient (includingcombinations thereof).

The cytokines in the pharmaceutical composition of the present inventionmay also include mediators known to be involved in the maintenance ofearly and late hematopoiesis such as IL-1 alpha and IL-1 beta, IL-6,IL-7, IL-8, IL-11 and IL-13; colony-stimulating factors, thrombopoietin,erythropoietin, stem cell factor, fit 3-ligand, hepatocyte cell growthfactor, tumor necrosis factor alpha, leukemia inhibitory factor,transforming growth factors beta 1 and beta 3; and macrophageinflammatory protein 1 alpha), angiogenic factors (fibroblast growthfactors 1 and 2, vascular endothelial growth factor) and mediators whoseusual target (and source) is the connective tissue-forming cells(platelet-derived growth factor A, epidermal growth factor, transforminggrowth factors alpha and beta 2, oncostatin M and insulin-like growthfactor-1), or neuronal cells (nerve growth factor) (Sensebe, L., et al.,Stem Cells 1997; 15:133-43), VEGF polypeptides that are present inplatelets and megacaryocytes (Wartiovaara, U., et al., Thromb Haemost1998; 80:171-5; Mohle, R., Proc Natl Acad Sci USA 1997; 94:663-8) HIF-1,a potent transcription factor that binds to and stimulates the promoterof several genes involved in responses to hypoxia, endothelial PASdomain protein 1 (EPAS 1), monocyte-derived cytokines for enhancingcollateral function such as monocyte chemotactic protein-1 (MCP-1).

In a preferred aspect, the pharmaceutical composition of the presentinvention is delivered via injection. These routes for administration(delivery) include, but are not limited to subcutaneous or parenteralincluding intravenous, intraarterial, intramuscular, intraperitoneal,intramyocardial, transendocardial, trans-epicardial, intranasaladministration as well as intrathecal, and infusion techniques. Hence,preferably the pharmaceutical composition is in a form that is suitablefor injection.

When administering a therapeutic of the present invention parenterally,it will generally be formulated in a unit dosage injectable form(solution, suspension, emulsion). The pharmaceutical formulationssuitable for injection include sterile aqueous solutions or dispersionsand sterile powders for reconstitution into sterile injectable solutionsor dispersions. The carrier can be a solvent or dispersing mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like), suitablemixtures thereof, and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compound compositions

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. According tothe present invention, however, any vehicle, diluent, or additive usedwould have to be compatible with the compounds.

Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various amounts of the otheringredients, as desired.

The pharmaceutical composition of the present invention, e.g.,comprising a therapeutic compound, can be administered to the patient inan injectable formulation containing any compatible carrier, such asvarious vehicles, adjuvants, additives, and diluents; or the compoundsutilized in the present invention can be administered parenterally tothe patient in the form of slow-release subcutaneous implants ortargeted delivery systems such as monoclonal antibodies, iontophoretic,polymer matrices, liposomes, and microspheres.

The pharmaceutical composition utilized in the present invention can beadministered orally to the patient. Conventional methods such asadministering the compounds in tablets, suspensions, solutions,emulsions, capsules, powders, syrups and the like are usable. Knowntechniques which deliver the compound orally or intravenously and retainthe biological activity are preferred.

In one embodiment, a composition of the present invention can beadministered initially, and thereafter maintained by furtheradministration. For instance, a composition of the invention can beadministered in one type of composition and thereafter furtheradministered in a different or the same type of composition. Forexample, a composition of the invention can be administered byintravenous injection to bring blood levels to a suitable level. Thepatient's levels are then maintained by an oral dosage form, althoughother forms of administration, dependent upon the patient's condition,can be used.

It is noted that humans are treated generally longer than the mice orother experimental animals which treatment has a length proportional tothe length of the disease process and drug effectiveness. The doses maybe single doses or multiple doses over a period of several days, butsingle doses are preferred. Thus, one can scale up from animalexperiments, e.g., rats, mice, and the like, to humans, by techniquesfrom this disclosure and documents cited herein and the knowledge in theart, without undue experimentation.

The treatment generally has a length proportional to the length of thedisease process and drug effectiveness and the patient being treated.

The quantity of the pharmaceutical composition to be administered willvary for the patient being treated. In a preferred embodiment,2×10⁴-1×10⁵ stem cells and 50-500 μg/kg per day of a cytokine wereadministered to the patient. While there would be an obvious sizedifference between the hearts of a mouse and a human, it is possiblethat 2×10⁴-1×10⁵ stem cells would be sufficient in a human as well.However, the precise determination of what would be considered aneffective dose may be based on factors individual to each patient,including their size, age, size of the infarct, and amount of time sincedamage. Therefore, dosages can be readily ascertained by those skilledin the art from this disclosure and the knowledge in the art. Thus, theskilled artisan can readily determine the amount of compound andoptional additives, vehicles, and/or carrier in compositions and to beadministered in methods of the invention. Typically, any additives (inaddition to the active stem cell(s) and/or cytokine(s)) are present inan amount of 0.001 to 50 wt % solution in phosphate buffered saline, andthe active ingredient is present in the order of micrograms tomilligrams, such as about 0.0001 to about 5 wt %, preferably about0.0001 to about 1 wt %, most preferably about 0.0001 to about 0.05 wt %or about 0.001 to about 20 wt %, preferably about 0.01 to about 10 wt %,and most preferably about 0.05 to about 5 wt %. Of course, for anycomposition to be administered to an animal or human, and for anyparticular method of administration, it is preferred to determinetherefore: toxicity, such as by determining the lethal dose (LD) andLD₅₀ in a suitable animal model e.g., rodent such as mouse; and, thedosage of the composition(s), concentration of components therein andtiming of administering the composition(s), which elicit a suitableresponse. Such determinations do not require undue experimentation fromthe knowledge of the skilled artisan, this disclosure and the documentscited herein. And, the time for sequential administrations can beascertained without undue experimentation.

Examples of compositions comprising a therapeutic of the inventioninclude liquid preparations for orifice, e.g., oral, nasal, anal,vaginal, peroral, intragastric, mucosal (e.g., perlingual, alveolar,gingival, olfactory or respiratory mucosa) etc., administration such assuspensions, syrups or elixirs; and, preparations for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration), such as sterile suspensions oremulsions. Such compositions may be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose or the like. The compositions can also be lyophilized.The compositions can contain auxiliary substances such as wetting oremulsifying agents, pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, colors, and the like,depending upon the route of administration and the preparation desired.Standard texts, such as “REMINGTON'S PHARMACEUTICAL SCIENCE”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation.

Compositions of the invention, are conveniently provided as liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsionsor viscous compositions which may be buffered to a selected pH. Ifdigestive tract absorption is preferred, compositions of the inventioncan be in the “solid” form of pills, tablets, capsules, caplets and thelike, including “solid” preparations which are time-released or whichhave a liquid filling, e.g., gelatin covered liquid, whereby the gelatinis dissolved in the stomach for delivery to the gut. If nasal orrespiratory (mucosal) administration is desired, compositions may be ina form and dispensed by a squeeze spray dispenser, pump dispenser oraerosol dispenser. Aerosols are usually under pressure by means of ahydrocarbon. Pump dispensers can preferably dispense a metered dose or,a dose having a particular particle size.

Compositions of the invention can contain pharmaceutically acceptableflavors and/or colors for rendering them more appealing, especially ifthey are administered orally. The viscous compositions may be in theform of gels, lotions, ointments, creams and the like (e.g., fortransdermal administration) and will typically contain a sufficientamount of a thickening agent so that the viscosity is from about 2500 to6500 cps, although more viscous compositions, even up to 10,000 cps maybe employed. Viscous compositions have a viscosity preferably of 2500 to5000 cps, since above that range they become more difficult toadminister. However, above that range, the compositions can approachsolid or gelatin forms which are then easily administered as a swallowedpill for oral ingestion.

Liquid preparations are normally easier to prepare than gels, otherviscous compositions, and solid compositions. Additionally, liquidcompositions are somewhat more convenient to administer, especially byinjection or orally. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with mucosa, such as the lining of the stomach or nasalmucosa.

Obviously, the choice of suitable carriers and other additives willdepend on the exact route of administration and the nature of theparticular dosage form, e.g., liquid dosage form (e.g., whether thecomposition is to be formulated into a solution, a suspension, gel oranother liquid form), or solid dosage form (e.g., whether thecomposition is to be formulated into a pill, tablet, capsule, caplet,time release form or liquid-filled form).

Solutions, suspensions and gels normally contain a major amount of water(preferably purified water) in addition to the active compound. Minoramounts of other ingredients such as pH adjusters (e.g., a base such asNaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents, jelling agents, (e.g., methylcellulose),colors and/or flavors may also be present. The compositions can beisotonic, i.e., they can have the same osmotic pressure as blood andlacrimal fluid.

The desired isotonicity of the compositions of this invention may beaccomplished using sodium chloride, or other pharmaceutically acceptableagents such as dextrose, boric acid, sodium tartrate, propylene glycolor other inorganic or organic solutes. Sodium chloride is preferredparticularly for buffers containing sodium ions.

Viscosity of the compositions may be maintained at the selected levelusing a pharmaceutically acceptable thickening agent. Methylcellulose ispreferred because it is readily and economically available and is easyto work with. Other suitable thickening agents include, for example,xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer,and the like. The preferred concentration of the thickener will dependupon the agent selected. The important point is to use an amount whichwill achieve the selected viscosity. Viscous compositions are normallyprepared from solutions by the addition of such thickening agents.

A pharmaceutically acceptable preservative can be employed to increasethe shelf-life of the compositions. Benzyl alcohol may be suitable,although a variety of preservatives including, for example, parabens,thimerosal, chlorobutanol, or benzalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

Those skilled in the art will recognize that the components of thecompositions should be selected to be chemically inert with respect tothe active compound. This will present no problem to those skilled inchemical and pharmaceutical principles, or problems can be readilyavoided by reference to standard texts or by simple experiments (notinvolving undue experimentation), from this disclosure and the documentscited herein.

The inventive compositions of this invention are prepared by mixing theingredients following generally accepted procedures. For example theselected components may be simply mixed in a blender, or other standarddevice to produce a concentrated mixture which may then be adjusted tothe final concentration and viscosity by the addition of water orthickening agent and possibly a buffer to control pH or an additionalsolute to control tonicity. Generally the pH may be from about 3 to 7.5.Compositions can be administered in dosages and by techniques well knownto those skilled in the medical and veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular patient, and the composition form used for administration(e.g., solid vs. liquid). Dosages for humans or other mammals can bedetermined without undue experimentation by the skilled artisan, fromthis disclosure, the documents cited herein, and the knowledge in theart.

Suitable regimes for initial administration and further doses or forsequential administrations also are variable, may include an initialadministration followed by subsequent administrations; but nonetheless,may be ascertained by the skilled artisan, from this disclosure, thedocuments cited herein, and the knowledge in the art.

The pharmaceutical compositions of the present invention are used totreat cardiovascular diseases, including, but not limited to,atherosclerosis, ischemia, hypertension, restenosis, angina pectoris,rheumatic heart disease, congenital cardiovascular defects and arterialinflammation and other diseases of the arteries, arterioles andcapillaries or related complaint. Accordingly, the invention involvesthe administration of stem cells as herein discussed, alone or incombination with one or more cytokine, as herein discussed, for thetreatment or prevention of any one or more of these conditions or otherconditions involving weakness in the heart, as well as compositions forsuch treatment or prevention, use of stem cells as herein discussed,alone or in combination with one or more cytokine, as herein discussed,for formulating such compositions, and kits involving stem cells asherein discussed, alone or in combination with one or more cytokine, asherein discussed, for preparing such compositions and/or for suchtreatment, or prevention. And, advantageous routes of administrationinvolves those best suited for treating these conditions, such as viainjection, including, but are not limited to subcutaneous or parenteralincluding intravenous, intraarterial, intramuscular, intraperitoneal,intramyocardial, transendocardial, trans-epicardial, intranasaladministration as well as intrathecal, and infusion techniques.

The pharmaceutical compositions of the present invention may be used astherapeutic agents—i.e. in therapy applications. As herein, the terms“treatment” and “therapy” include curative effects, alleviation effects,and prophylactic effects.

As used herein, “patient” may encompass any vertebrate including but notlimited to humans, mammals, reptiles, amphibians and fish. However,advantageously, the patient is a mammal such as a human, or an animalmammal such as a domesticated mammal, e.g., dog, cat, horse, and thelike, or production mammal, e.g., cow, sheep, pig, and the like

As used herein “somatic stem cell” or “stem cell” or “hematopoieticcell” refers to either autologous or allogenic stem cells, which may beobtained from the bone marrow, peripheral blood, or other source.

As used herein, “adult” stem cells refers to stem cells that are notembryonic in origin nor derived from embryos or fetal tissue.

As used herein “recently damaged myocardium” refers to myocardium whichhas been damaged within one week of treatment being started. In apreferred embodiment, the myocardium has been damaged within three daysof the start of treatment. In a further preferred embodiment, themyocardium has been damaged within 12 hours of the start of treatment.It is advantageous to employ stem cells alone or in combination withcytokine(s) as herein disclosed to a recently damaged myocardium.

As used herein “damaged myocardium” refers to myocardial cells whichhave been exposed to ischemic conditions. These ischemic conditions maybe caused by a myocardial infarction, or other cardiovascular disease orrelated complaint. The lack of oxygen causes the death of the cells inthe surrounding area, leaving an infarct, which will eventually scar.

As used herein, “home” refers to the attraction and mobilization ofsomatic stem cells towards damaged myocardium and/or myocardial cells.

As used herein, “assemble” refers to the assembly of differentiatedsomatic stem cells into functional structures i.e., myocardium and/ormyocardial cells, coronary arteries, arterioles, and capillaries etc.This assembly provides functionality to the differentiated myocardiumand/or myocardial cells, coronary arteries, arterioles and capillaries.

Thus, the invention involves the use of somatic stem cells. These arepresent in animals in small amounts, but methods of collecting stemcells are known to those skilled in the art.

In another aspect of the invention, the stem cells are selected to belineage negative. The term “lineage negative” is known to one skilled inthe art as meaning the cell does not express antigens characteristic ofspecific cell lineages.

Advantageously, the lineage negative stem cells are selected to be c-kitpositive. The term “c-kit” is known to one skilled in the art as being areceptor which is known to be present on the surface of stem cells, andwhich is routinely utilized in the process of identifying and separatingstem cells from other surrounding cells.

The invention further involves a therapeutically effective dose oramount of stem cells applied to the heart. An effective dose is anamount sufficient to effect a beneficial or desired clinical result.Said dose could be administered in one or more administrations. In theexamples that follow, 2×10⁴-10⁵ stem cells were administered in themouse model. While there would be an obvious size difference between thehearts of a mouse and a human, it is possible that this range of stemcells would be sufficient in a human as well. However, the precisedetermination of what would be considered an effective dose may be basedon factors individual to each patient, including their size, age, sizeof the infarct, and amount of time since damage. One skilled in the art,specifically a physician or cardiologist, would be able to determine thenumber of stem cells that would constitute an effective dose withoutundue experimentation.

In another aspect of the invention, the stem cells are delivered to theheart, specifically to the border area of the infarct. As one skilled inthe art would be aware, the infarcted area is visible grossly, allowingthis specific placement of stem cells to be possible.

The stem cells are advantageously administered by injection,specifically an intramyocardial injection. As one skilled in the artwould be aware, this is the preferred method of delivery for stem cellsas the heart is a functioning muscle. Injection of the stem cells intothe heart ensures that they will not be lost due to the contractingmovements of the heart.

In a further aspect of the invention, the stem cells are administered byinjection transendocardially or trans-epicardially. This preferredembodiment allows the stem cells to penetrate the protective surroundingmembrane, necessitated by the embodiment in which the cells are injectedintramyocardially.

A preferred embodiment of the invention includes use of a catheter-basedapproach to deliver the trans-endocardial injection. The use of acatheter precludes more invasive methods of delivery wherein the openingof the chest cavity would be necessitated. As one skilled in the art isaware, optimum time of recovery would be allowed by the more minimallyinvasive procedure, which as outlined here, includes a catheterapproach.

Further embodiments of the invention require the stem cells to migrateinto the infarcted region and differentiate into myocytes, smooth musclecells, and endothelial cells. It is known in the art that these types ofcells must be present to restore both structural and functionalintegrity. Other approaches to repairing infarcted or ischemic tissuehave involved the implantation of these cells directly into the heart,or as cultured grafts, such as in U.S. Pat. Nos. 6,110,459, and6,099,832.

Another embodiment of the invention includes the proliferation of thedifferentiated cells and the formation of the cells into cardiacstructures including coronary arteries, arterioles, capillaries, andmyocardium. As one skilled in the art is aware, all of these structuresare essential for proper function in the heart. It has been shown in theliterature that implantation of cells including endothelial cells andsmooth muscle cells will allow for the implanted cells to live withinthe infarcted region, however they do not form the necessary structuresto enable the heart to regain full functionality. The ability to restoreboth functional and structural integrity is yet another aspect of thisinvention.

Another aspect of the invention relates to the administration of acytokine. This cytokine may be chosen from a group of cytokines, or mayinclude combinations of cytokines. Stem cell factor (SCF) andgranulocyte-colony stimulating factor (G-CSF) are known by those skilledin the art as stimulating factors which cause the mobilization of stemcells into the blood stream (Bianco et al, 2001, Clutterbuck, 1997,Kronenwett et al, 2000, LaIuppa et al, 1997, Patchen et al, 1998).Stromal cell-derived factor-1 has been shown to stimulate stem cellmobilization chemotactically, while steel factor has both chemotacticand chemokinetic properties (Caceres-Cortes et al, 2001, Jo et al, 2000,Kim and Broxmeyer, 1998, Ikuta et al, 1991). Vascular endothelial growthfactor has been surmised to engage a paracrine loop that helpsfacilitate migration during mobilization (Bautz et al, 2000,Janowska-Wieczorek et al, 2001). Macrophage colony stimulating factorand granulocyte-macrophage stimulating factor have been shown tofunction in the same manner of SCF and G-CSF, by stimulatingmobilization of stem cells. Interleukin-3 has also been shown tostimulate mobilization of stem cells, and is especially potent incombination with other cytokines.

The cytokine can be administered via a vector that expresses thecytokine in vivo. A vector for in vivo expression can be a vector orcells or an expression system as cited in any document incorporatedherein by reference or used in the art, such as a viral vector, e.g., anadenovirus, poxvirus (such as vaccinia, canarypox virus, MVA, NYVAC,ALVAC, and the like), lentivirus or a DNA plasmid vector; and, thecytokine can also be from in vitro expression via such a vector or cellsor expression system or others such as a baculovirus expression system,bacterial vectors such as E. coli, and mammalian cells such as CHOcells. See, e.g., U.S. Pat. Nos. 6,265,189, 6,130,066, 6,004,777,5,990,091, 5,942,235, 5,833,975. The cytokine compositions may lendthemselves to administration by routes outside of those stated to beadvantageous or preferred for stem cell preparations; but, cytokinecompositions may also be advantageously administered by routes stated tobe advantageous or preferred for stem cell preparations.

A further aspect of the invention involves administration of atherapeutically effective dose or amount of a cytokine. An effectivedose is an amount sufficient to effect a beneficial or desired clinicalresult. Said dose could be administered in one or more administrations.In a preferred embodiment, the dose would be given over the course ofabout two or three days following the beginning of treatment. However,the precise determination of what would be considered an effective dosemay be based on factors individual to each patient, including theirsize, age, size of the infarct, the cytokine or combination of cytokinesbeing administered, and amount of time since damage. One skilled in theart, specifically a physician or cardiologist, would be able todetermine a sufficient amount of cytokine that would constitute aneffective dose without being subjected to undue experimentation.

The invention also involves the administration of the therapeuticallyeffective dose or amount of a cytokine being delivered by injection,specifically subcutaneously or intravenously. A person skilled in theart will be aware that subcutaneous injection or intravenous deliveryare extremely common and offer an effective method of delivering thespecific dose in a manner which allows for timely uptake and circulationin the blood stream.

A further aspect of the invention includes the administered cytokinestimulating the patient's stem cells and causing mobilization into theblood stream. As mentioned previously, the given cytokines arewell-known to one skilled in the art for their ability to promote saidmobilization.

Advantageously, once the stem cells have mobilized into the bloodstream,they home to the damaged area of the heart, as will become clear throughthe following examples.

Further embodiments of the invention involve the stem cells migratinginto the infarcted region and differentiating into myocytes, smoothmuscle cells, and endothelial cells. It is known in the art that thesetypes of cells must be present to restore both structural and functionalintegrity.

Another embodiment of the invention includes the proliferation of thedifferentiated cells and the formation of the cells into cardiacstructures including coronary arteries, arterioles, capillaries, andmyocardium. As one skilled in the art is aware, all of these structuresare important for proper function in the heart. It has been shown in theliterature that implantation of cells including endothelial cells andsmooth muscle cells will allow for the implanted cells to live withinthe infarcted region, however they do not form the necessary structuresto enable the heart to regain full functionality. The ability to restoreboth functional and structural integrity or better functional andstructural integrity than previously achieved in the art is yet anotheraspect of this invention.

It is a preferred in the practice of the invention to utilize both theadministration of stem cells and that of a cytokine to ensure the mosteffective method of repairing damaged myocardium.

Stem cells employed in the invention are advantageously selected to belineage negative. The term “lineage negative” is known to one skilled inthe art as meaning the cell does not express antigens characteristic ofspecific cell lineages. And, it is advantageous that the lineagenegative stem cells are selected to be c-kit positive. The term “c-kit”is known to one skilled in the art as being a receptor which is known tobe present on the surface of stem cells, and which is routinely utilizedin the process of identifying and separating stem cells from othersurrounding cells.

In certain embodiments, a therapeutically effective dose of stem cellsis applied, delivered, or administered to the heart or implanted intothe heart. An effective dose or amount is an amount sufficient to effecta beneficial or desired clinical result. Said dose could be administeredin one or more administrations. In the examples that follow, 2×10⁴-1×10⁵stem cells were administered in the mouse model. While there would be anobvious size difference between the hearts of a mouse and a human, it ispossible that 2×10⁴-1×10⁵ stem cells would be sufficient in a human aswell. However, the precise determination of what would be considered aneffective dose may be based on factors individual to each patient,including their size, age, size of the infarct, and amount of time sincedamage. One skilled in the art, specifically a physician orcardiologist, would be able to determine the number and type (or types)of stem cells which would constitute an effective dose without beingsubjected to undue experimentation, from this disclosure and theknowledge in the art; and, in this regard and in general in regard topreparing formulations and administering formulations or componentsthereof, mention is made of the teachings in the Examples and that theskilled artisan can scale dosages, amounts and the like based on theweight of the patient to be treated in comparison to the weight of anyanimal employed in the Examples. The stem cells are advantageously bonemarrow or are cardiac stem cells; and even more advantageously, the stemcells are adult bone marrow (hematopoietic stem cells) or adult cardiacstem cells or a combination thereof or a combination of cardiac stemcells such as adult cardiac stem cells and another type of stem cellsuch as another type of adult stem cells.

In another aspect of the invention, the stem cells are delivered to theheart, specifically to the border area of the infarct. As one skilled inthe art would be aware, the infarcted area is visible grossly, allowingthis specific placement of stem cells to be possible.

The stem cells are advantageously administered by injection,specifically an intramyocardial injection. As one skilled in the artwould be aware, this is the preferred method of delivery for stem cellsas the heart is a functioning muscle. Injection of the stem cells intothe heart ensures that they will not be lost due to the contractingmovements of the heart.

In other aspects of the invention, the stem cells are administered byinjection transendocardially or trans-epicardially. This preferredembodiment allows the stem cells to penetrate the protective surroundingmembrane, necessitated by the embodiment in which the cells are injectedintramyocardially.

A preferred embodiment of the invention includes use of a catheter-basedapproach to deliver the trans-endocardial injection. The use of acatheter precludes more invasive methods of delivery wherein the openingof the chest cavity would be necessitated. As one skilled in the art isaware, optimum time of recovery would be allowed by the more minimallyinvasive procedure, which as outlined here, includes a catheterapproach.

Embodiments of the invention can involve the administration of acytokine. This cytokine may be chosen from a group of cytokines, or mayinclude combinations of cytokines.

A further aspect of the invention involves administration of atherapeutically effective dose of a cytokine. An effective dose oramount is an amount sufficient to effect a beneficial or desiredclinical result. Said dose could be administered in one or moreadministrations. In a preferred embodiment, the dose would be given overthe course of about two or three days following the beginning oftreatment. However, the precise determination of what would beconsidered an effective dose may be based on factors individual to eachpatient, including their size, age, size of the infarct, the cytokine orcombination of cytokines being administered, and amount of time sincedamage. One skilled in the art, specifically a physician orcardiologist, would be able to determine a sufficient amount of cytokinethat would constitute an effective dose without being subjected to undueexperimentation, especially in view of the disclosure herein and theknowledge in the art.

The administration of the therapeutically effective dose of at least onecytokine is advantageously by injection, specifically subcutaneously orintravenously. A person skilled in the art will be aware thatsubcutaneous injection or intravenous delivery are extremely common andoffer an effective method of delivering the specific dose in a mannerwhich allows for timely uptake and circulation in the blood stream.

A further aspect of the invention includes the administered cytokinestimulating the patient's stem cells and causing mobilization into theblood stream. As mentioned previously, the given cytokines are wellknown to one skilled in the art for their ability to promote saidmobilization. Again, once the stem cells have mobilized into thebloodstream, they home to the damaged area of the heart. Thus in certainembodiments, both the implanted stem cells and the mobilized stem cellsmigrate into the infarct region and differentiate into myocytes, smoothmuscle cells, and endothelial cells. It is known in the art that thesetypes of cells are advantageously present to restore both structural andfunctional integrity.

Another embodiment of the invention includes the proliferation of thedifferentiated cells and the formation of the cells into cardiacstructures including coronary arteries, arterioles, capillaries, andmyocardium. As one skilled in the art is aware, all of these structuresare essential for proper function in the heart. It has been shown in theliterature that implantation of cells including endothelial cells andsmooth muscle cells will allow for the implanted cells to live withinthe infarcted region, however they do not form the necessary structuresto enable the heart to regain full functionality. Cardiac structures canbe generated ex vivo and then implanted in the form of a graft; with theimplantation of the graft being alone or in combination with stem cellsor stem cells and at least one cytokine as in this disclosure, e.g.,advantageously adult or cardiac or hematopoietic stem cells such asadult cardiac and/or adult hematopoietic stem cells or adult cardiacstem cells with another type of stem cell e.g. another type of adultstem cell. The means of generating and/or regenerating myocardium exvivo, may incorporate somatic stem cells and heart tissue being culturedin vitro, optionally in the presence of a cytokine. The somatic stemcells differentiate into myocytes, smooth muscle cells and endothelialcells, and proliferate in vitro, forming myocardial tissue and/or cells.These tissues and cells may assemble into cardiac structures includingarteries, arterioles, capillaries, and myocardium. The tissue and/orcells formed in vitro may then be implanted into a patient, e.g. via agraft, to restore structural and functional integrity.

Additionally or alternatively, the source of the tissue being graftedcan be from other sources of tissue used in grafts of the heart.

The restoration or some restoration of both functional and structuralintegrity of cardiac tissue—advantageously over that which has occurredpreviously—is yet another aspect of this invention.

Accordingly, the invention comprehends, in further aspects, methods forpreparing compositions such as pharmaceutical compositions includingsomatic stem cells and/or at least one cytokine, for instance, for usein inventive methods for treating cardiovascular disease or conditionsor cardiac conditions.

The present invention is additionally described by way of the following,non-limiting examples, that provide a better understanding of thepresent invention and of its many advantages.

All of the materials, reagents, chemicals, assays, cytokines,antibodies, and miscellaneous items referred to in the followingexamples are readily available to the research community throughcommercial suppliers, including but not limited to, Genzyme, Invitrogen,Gibco BRL, Clonetics, Fisher Scientific, R & D Systems, MBLInternational Corporation, CN Biosciences Corporate, Sigma Aldrich, andCedarLane Laboratories, Limited.

For example,

-   -   stem cell factor is available under the name SCF (multiple forms        of recombinant human, recombinant mouse, and antibodies to        each), from R & D Systems (614 McKinley Place N.E., Minneapolis,        Minn. 55413);    -   granulocyte-colony stimulating factor is available under the        name G-CSF (multiple forms of recombinant human, recombinant        mouse, and antibodies to each), from R & D Systems;    -   stem cell antibody-1 is available under the name SCA-1 from MBL        International Corporation (200 Dexter Avenue, Suite D,        Watertown, Mass. 02472);    -   multidrug resistant antibody is available under the name        Anti-MDR from CN Biosciences Corporate;    -   c-kit antibody is available under the name c-kit (Ab-1)        Polyclonal Antibody from CN Biosciences Corporate (Affiliate of        Merck KgaA, Darmstadt, Germany. Corporate headquarters located        at 10394 Pacific Center Court, San Diego, Calif. 92121).

EXAMPLES Example 1 Hematopoietic Stem Cell (HSC) Repair of InfarctedMyocardium

A. Harvesting of Hematopoietic Stem Cells

Bone marrow was harvested from the femurs and tibias of male transgenicmice expressing enhanced green fluorescent protein (EGFP). Aftersurgical removal of the femurs and tibias, the muscle was dissected andthe upper and lower surface of the bone was cut on the surface to allowthe collecting buffer to infiltrate the bone marrow. The fluidcontaining buffer and cells was collected in tubes such as 1.5 mlEpindorf tubes. Bone marrow cells were suspended in PBS containing 5%fetal calf serum (FCS) and incubated on ice with rat anti-mousemonoclonal antibodies specific for the following hematopoietic lineages:CD4 and CD8 (T-lymphocytes), B-220 (B-lymphocytes), Mac-1 (macrophages),GR-1 (granulocytes) (Caltag Laboratories) and TER-119 (erythrocytes)(Pharmingen). Cells were then rinsed in PBS and incubated for 30 minuteswith magnetic beads coated with goat anti-rat immunoglobulin(Polysciences Inc.). Lineage positive cells (Lin⁺) were removed by abiomagnet and lineage negative cells (Lin⁻) were stained withACK-4-biotin (anti-c-kit mAb). Cells were rinsed in PBS, stained withstreptavidin-conjugated phycoerythrin (SA-PE) (Caltag Labs.) and sortedby fluorescence activated cell sorting (FACS) using a FACSVantageinstrument (Becton Dickinson). Excitation of EGFP and ACK-4-biotin-SA-EPoccurred at a wavelength of 488 nm. The Lin⁻ cells were sorted as c-kitpositive (c-kit^(pos)) and c-kit negative (c-kit^(NEG)) with a 1-2 logdifference in staining intensity (FIG. 1). The c-kit^(POS) cells weresuspended at 2×10⁴ to 1×10⁵ cells in 5 μl of PBS and the c-kit^(NEG)cells were suspended at a concentration of 1×10⁵ in 5 μl of PBS.

B. Induction of Myocardial Infarction in Mice

Myocardial infarction was induced in female C57BL/6 mice at 2 months ofage as described by Li et al. (1997). Three to five hours afterinfarction, the thorax of the mice was reopened and 2.5 μl of PBScontaining Lin⁻c-kit^(POS) cells were injected in the anterior andposterior aspects of the viable myocardium bordering the infarct (FIG.2). Infarcted mice, left uninjected or injected with Lin⁻c-kit-^(NEG)cells, and sham-operated mice i.e., mice where the chest cavity wasopened but no infarction was induced, were used as controls. All animalswere sacrificed 9±2 days after surgery. Protocols were approved byinstitutional review board. Results are presented as mean ±SD.Significance between two measurements was determined by the Student's ttest, and in multiple comparisons was evaluated by the Bonferroni method(Scholzen and Gerdes, 2000). P<0.05 was considered significant.

Injection of male Lin⁻c-kit^(POS) bone marrow cells in theperi-infarcted left ventricle of female mice resulted in myocardialregeneration. The peri-infarcted region is the region of viablemyocardium bordering the infarct. Repair was obtained in 12 of 30 mice(40%). Failure to reconstitute infarcts was attributed to the difficultyof transplanting cells into tissue contracting at 600 beats per minute(bpm). However, an immunologic reaction to the histocompatibilityantigen on the Y chromosome of the donor bone marrow cells could accountfor the lack of repair in some of the female recipients. Closely packedmyocytes occupied 68±11% of the infarcted region and extended from theanterior to the posterior aspect of the ventricle (FIGS. 2A-2D). Newmyocytes were not found in mice injected with Lin⁻c-kit^(NEG) cells(FIG. 2E).

C. Determination of Ventricular Function

Mice were anesthetized with chloral hydrate (400 mg/kg body weight,i.p.), and the night carotid artery was cannulated with a microtippressure transducer (model SPR-671, Millar) for the measurements of leftventricular (LV) pressures and LV+ and −dP/dt in the closed-chestpreparation to determine whether developing myocytes derived from theHSC transplant had an impact on function. Infarcted mice non-injected orinjected with Lin⁻c-kit^(NEG) cells were combined in the statistics. Incomparison with sham-operated groups, the infarcted groups exhibitedindices of cardiac failure (FIG. 3). In mice treated withLin-c-kit^(POS) cells, LV end-diastolic pressure (LVEDP) was 36% lower,and developed pressure (LVDP) and LV+ and −dP/dt were 32%, 40%, and 41%higher, respectively (FIG. 4A).

D. Determination of Cell Proliferation and EGFP Detection

The abdominal aorta was cannulated, the heart was arrested in diastoleby injection of cadmium chloride (CdCl₂), and the myocardium wasperfused retrogradedly with 10% buffered formalin. Three tissuesections, from the base to the apex of the left ventricle, were stainedwith hematoxylin and eosin. At 9±2 days after coronary occlusion, theinfarcted portion of the ventricle was easily identifiable grossly andhistologically (see FIG. 2A). The lengths of the endocardial andepicardial surfaces delimiting the infarcted region, and the endocardiumand epicardium of the entire left ventricle were measured in eachsection. Subsequently, their quotients were computed to yield theaverage infarct size in each case. This was accomplished at 4×magnification utilizing an image analyzer connected to a microscope. Thefraction of endocardial and epicardial circumference delimiting theinfarcted area (Pfeffer and Braunwald, 1990; Li et al., 1997) did notdiffer in untreated mice, 78±18% (n=8) and in mice treated withLin⁻c-kit^(POS) cells (n=12), 75±14% or Lin⁻c-kit^(NEG) cells (n=11),75±15%.

To establish whether Lin⁻c-kit^(POS) cells resulted in myocardialregeneration, BrdU (50 mg/kg body weight, i.p.) was administered dailyto the animals for 4-5 consecutive days before sacrifice to determinecumulative cell division during active growth. Sections were incubatedwith anti-BrdU antibody and BrdU labeling of cardiac cell nuclei in theS phase was measured. Moreover, expression of Ki67 in nuclei (Ki67 isexpressed in cycling cells in G1, S, G2, and early mitosis) wasevaluated by treating samples with a rabbit polyclonal anti-mouse Ki67antibody (Dako Corp.). FITC-conjugated goat anti-rabbit IgG was used assecondary antibody. (FIGS. 5 and 6). EGFP was detected with a rabbitpolyclonal anti-GFP (Molecular Probes). Myocytes were recognized with amouse monoclonal anti-cardiac myosin heavy chain (MAB 1548; Chemicon) ora mouse monoclonal anti-α-sarcomeric actin (clone 5C5; Sigma),endothelial cells with a rabbit polyclonal anti-human factor VIII(Sigma) and smooth muscle cells with a mouse monoclonal anti-α-smoothmuscle actin (clone 1A4; Sigma). Nuclei were stained with propidiumiodide (PI), 10 μg/ml. The percentages of myocyte (M), endothelial cell(EC) and smooth muscle cell (SMC) nuclei labeled by BrdU and Ki67 wereobtained by confocal microscopy. This was accomplished by dividing thenumber of nuclei labeled by the total number of nuclei examined. Numberof nuclei sampled in each cell population was as follows; BrdU labeling:M=2,908; EC=2,153; SMC=4,877. Ki67 labeling: M=3,771; EC=4,051;SMC=4,752. Number of cells counted for EGFP labeling: M=3,278; EC=2,056;SMC=1,274. The percentage of myocytes in the regenerating myocardium wasdetermined by delineating the area occupied by cardiac myosin stainedcells divided by the total area represented by the infarcted region ineach case. Myocyte proliferation was 93% (p<0.001) and 60% (p<0.001)higher than in endothelial cells, and 225% (p<0.001 and 176% (p<0.001)higher than smooth muscle cells, when measured by BrdU and Ki67,respectively.

The origin of the cells in the forming myocardium was determined by theexpression of EGFP (FIGS. 7 and 8). EGFP expression was restricted tothe cytoplasm and the Y chromosome to nuclei of new cardiac cells. EGFPwas combined with labeling of proteins specific for myocytes,endothelial cells and smooth muscle cells. This allowed theidentification of each cardiac cell type and the recognition ofendothelial cells and smooth muscle cells organized in coronary vessels(FIGS. 5, 7, and 8). The percentage of new myocytes, endothelial cellsand smooth muscle cells that expressed EGFP was 53±9% (n=7), 44±6% (n=7)and 49±7% (n=7), respectively. These values were consistent with thefraction of transplanted Lin⁻c-kit^(POS) bone marrow cells thatexpressed EGFP, 44±10% (n=6). An average 54±8% (n=6) of myocytes,endothelial cells and smooth muscle cells expressed EGFP in the heart ofdonor transgenic mice.

E. Detection of the Y-Chromosome For the fluorescence in situhybridization (FISH) assay, sections were exposed to a denaturingsolution containing 70% formamide. After dehydration with ethanol,sections were hybridized with the DNA probe CEP Y (satellite III)Spectrum Green (Vysis) for 3 hours. Nuclei were stained with PI.

Y-chromosomes were not detected in cells from the surviving portion ofthe ventricle. However, the Y-chromosome was detected in the newlyformed myocytes, indicating their origin as from the injectedbone-marrow cells (FIG. 9).

F. Detection of Transcription Factors and Connexin 43

Sections were incubated with rabbit polyclonal anti-MEF2 (C-21; SantaCruz), rabbit polyclonal anti-GATA-4 (H-112; Santa Cruz), rabbitpolyclonal anti-Csx/NRx2.5 (obtained from Dr. Izumo) and rabbitpolyclonal anti-connexin 43 (Sigma). FITC-conjugated goat anti-rabbitIgG (Sigma) was used as secondary antibody.

To confirm that newly formed myocytes represented maturing cells aimingat functional competence, the expression of the myocyte enhancer factor2 (MEF2), the cardiac specific transcription factor GATA-4 and the earlymarker of myocyte development Csx/NRx2.5 was examined. In the heart,MEF2 proteins are recruited by GATA-4 to synergistically activate thepromoters of several cardiac genes such as myosin light chain, troponinT, troponin I, α-myosin heavy chain, desmin, atrial natriuretic factorand α-actin (Durocher et al., 1997; Morin et al., 2000). Csx/NRx2.5 is atranscription factor restricted to the initial phases of myocytedifferentiation (Durocher et al., 1997). In the reconstituting heart,all nuclei of cardiac myosin labeled cells expressed MEF2 (FIGS. 7D-7F)and GATA-4 (FIG. 10), but only 40±9% expressed Csx/NRx2.5 (FIGS. 7G-7I).To characterize further the properties of these myocytes, the expressionof connexin 43 was determined. This protein is responsible forintercellular connections and electrical coupling through the generationof plasma membrane channels between myocytes (Beardsle et al., 1998;Musil et al., 2000); connexin 43 was apparent in the cell cytoplasm andat the surface of closely aligned differentiating cells (FIGS. 11A-11D).These results were consistent with the expected functional competence ofthe heart muscle phenotype. Additionally, myocytes at various stages ofmaturation were detected within the same and different bands (FIG. 12).

Example 2 Mobilization of Bone Marrow Cells to Repair InfarctedMyocardium

A. Myocardial Infarction and Cytokines.

Fifteen C57BL/6 male mice at 2 months of age were splenectomized and 2weeks later were injected subcutaneously with recombinant rat stem cellfactor (SCF), 200 μg/kg/day, and recombinant human granulocyte colonystimulating factor (G-CSF), 50 μg/kg/day (Amgen), once a day for 5 days(Bodine et al., 1994; Orlic et al., 1993). Under ether anesthesia, theleft ventricle (LV) was exposed and the coronary artery was ligated(Orlic et al., 2001; Li et al., 1997; Li et al., 1999). SCF and G-CSFwere given for 3 more days. Controls consisted of splenectomizedinfarcted and sham-operated (SO) mice injected with saline. BrdU, 50mg/kg body weight, was given once a day, for 13 days, before sacrifice;mice were killed at 27 days. Protocols were approved by New York MedicalCollege. Results are mean ±SD. Significance was determined by theStudent's t test and Bonferroni method (Li et al., 1999). Mortality wascomputed with log-rank test. P<0.05 was significant.

Given the ability of bone marrow Lin⁻c-kit^(POS) cells totransdifferentiate into the cardiogenic lineage (Orlic et al., 2001), aprotocol was used to maximize their number in the peripheral circulationin order to increase the probability of their homing to the region ofdead myocardium. In normal animals, the frequency of Lin⁻c-kit^(POS)cells in the blood is only a small fraction of similar cells present inthe bone marrow (Bodine et al., 1994; Orlic et al., 1993). As documentedpreviously, the cytokine treatment used here promotes a marked increaseof Lin⁻c-kit^(POS) cells in the bone marrow and a redistribution ofthese cells from the bone marrow to the peripheral blood. This protocolleads to a 250-fold increase in Lin⁻c-kit^(POS) cells in the circulation(Bodine et al., 1994; Orlic et al., 1993).

In the current study, BMC mobilization by SCF and G-CSF resulted in adramatic increase in survival of infarcted mice; with cytokinetreatment, 73% of mice (11 of 15) survived 27 days, while mortality wasvery high in untreated infarcted mice (FIG. 13A). A large number ofanimals in this group died from 3 to 6 days after myocardial infarction(MI) and only 17% (9 of 52) reached 27 days (p<0.001). Mice that diedwithin 48 hours post-MI were not included in the mortality curve tominimize the influence of the surgical trauma. Infarct size was similarin the cytokine-, 64±11% (n=11), and saline-, 62±19% (n=9), injectedanimals as measured by the number of myocytes lost in the leftventricular free wall (LVFW) at 27 days (FIG. 14).

Importantly, bone marrow cell mobilization promoted myocardialregeneration in all 11 cytokine-treated infarcted mice, sacrificed 27days after surgery (FIG. 13B). Myocardial growth within the infarct wasalso seen in the 4 mice that died prematurely at day 6 (n=2) and at day9 (n=2). Cardiac repair was characterized by a band of newly formedmyocardium occupying most of the damaged area. The developing tissueextended from the border zone to the inside of the injured region andfrom the endocardium to the epicardium of the LVFW. In the absence ofcytokines, myocardial replacement was never observed and healing withscar formation was apparent (FIG. 13C). Conversely, only small areas ofcollagen accumulation were detected in treated mice.

B. Detection of BMC Mobilization by Echocardiography and Hemodynamics.

Echocardiography was performed in conscious mice using a Sequoia 256c(Acuson) equipped with a 13-MHz linear transducer (15L8). The anteriorchest area was shaved and two dimensional (2D) images and M-modetracings were recorded from the parasternal short axis view at the levelof papillary muscles. From M-mode tracings, anatomical parameters indiastole and systole were obtained (Pollick et al., 1995). Ejectionfraction (EF) was derived from LV cross sectional area in 2D short axisview (Pollick et al., 1995): EF=[(LVDA−LVSA)/LVDA]*100 where LVDA andLVSA correspond to LV areas in diastole and in systole. Mice wereanesthetized with chloral hydrate (400 mg/kg body weight, ip) and amicrotip pressure transducer (SPR-671, Millar) connected to a chartrecorder was advanced into the LV for the evaluation of pressures and +and −dP/dt in the closed-chest preparation (Orlic et al., 2001; Li etal., 1997; Li et al., 1999).

EF was 48%, 62% and 114% higher in treated than in non-treated mice at9, 16 and 26 days after coronary occlusion, respectively (FIG. 15D). Inmice exposed to cytokines, contractile function developed with time inthe infarcted region of the wall (FIGS. 15E-M; FIGS. 16H-P,www.pnas.org). Conversely, LV end-diastolic pressure (LVEDP) increased76% more in non-treated mice. The changes in LV systolic pressure (notshown), developed pressure (LVDP), + and −dP/dt were also more severe inthe absence of cytokine treatment (FIGS. 17A-D). Additionally, theincrease in diastolic stress in the zone bordering and remote frominfarction was 69-73% lower in cytokine-treated mice (FIG. 15N).Therefore, cytokine-mediated infarct repair restored a noticeable levelof contraction in the regenerating myocardium, decreasing diastolic wallstress and increasing ventricular performance. Myocardial regenerationattenuated cavitary dilation and mural thinning during the evolution ofthe infarcted heart in vivo.

Echocardiographically, LV end-systolic (LVESD) and end-diastolic (LVEDD)diameters increased more in non-treated than in cytokine-treated mice,at 9, 16 and 26 days after infarction (FIGS. 16A-B). Infarctionprevented the evaluation of systolic (AWST) and diastolic (AWDT)anterior wall thickness. When measurable, the posterior wall thicknessin systole (PWST) and diastole (PWDT) was greater in treated mice (FIGS.16C-D). Anatomically, the wall bordering and remote from infarction was26% and 22% thicker in cytokine-injected mice (FIG. 16E). BMC-inducedrepair resulted in a 42% higher wall thickness-to-chamber radius ratio(FIG. 15A). Additionally, tissue regeneration decreased the expansion incavitary diameter, −14%, longitudinal axis, −5% (FIGS. 16F-G), andchamber volume, −26% (FIG. 15B). Importantly, ventricularmass-to-chamber volume ratio was 36% higher in treated animals (FIG.15C). Therefore, BMC mobilization that led to proliferation anddifferentiation of a new population of myocytes and vascular structuresattenuated the anatomical variables which define cardiac decompensation.

C. Cardiac Anatomy and Determination of Infarct Size.

Following hemodynamic measurements, the abdominal aorta was cannulated,the heart was arrested in diastole with CdCl₂ and the myocardium wasperfused with 10% formalin. The LV chamber was filled with fixative at apressure equal to the in vivo measured end-diastolic pressure (Li etal., 1997; Li et al., 1999). The LV intracavitary axis was measured andthree transverse slices from the base, mid-region and apex were embeddedin paraffin. The mid-section was used to measure LV thickness, chamberdiameter and volume (Li et al., 1997; Li et al., 1999). Infarct size wasdetermined by the number of myocytes lost from the LVFW (Olivetti etal., 1991; Beltrami et al., 1994).

To quantify the contribution of the developing band to the ventricularmass, firstly the volume of the LVFW (weight divided by 1.06 g/ml) wasdetermined in each group of mice. The data was 56±2 mm³ in sham operated(SO), 62±4 mm³ (viable FW=41±3; infarcted FW=21±4) in infarctednon-treated animals, and 56±9 mm³ (viable FW=37±8; infarcted FW=19±5) ininfarcted cytokine-treated mice. These values were compared to theexpected values of spared [EXPLAIN] and lost myocardium at 27 days,given the size of the infarct in the non-treated and cytokine-treatedanimals. From the volume of the LVFW (56 mm³) in SO and infarct size innon-treated, 62%, and treated, 64%, mice, it was possible to calculatethe volume of myocardium destined to remain (non-treated=21 mm³;treated=20 mm³) and destined to be lost (non-treated=35 mm³; treated=36mm³) 27 days after coronary occlusion (FIG. 18A). The volume of newlyformed myocardium was detected exclusively in cytokine-treated mice andfound to be 14 mm³ (FIG. 18A). Thus, the repair band reduced infarctsize from 64% (36 mm³/56 mm³=64%) to 39% [(36 mm³−14 mm³)/56 mm³=39%].Since the spared portion of the LVFW at 27 days was 41 and 37 mm³ innon-treated and treated mice (see above), the remaining myocardium,shown in FIG. 18 a, underwent 95% (p<0.001) and 85% (p<0.001)hypertrophy, respectively. Consistently, myocyte cell volume increased94% and 77% (FIG. 18B).

D. Determination the Total Volume of Formed Myocardium

The volume of regenerating myocardium was determined by measuring ineach of three sections the area occupied by the restored tissue andsection thickness. The product of these two variables yielded the volumeof tissue repair in each section. Values in the three sections wereadded and the total volume of formed myocardium was obtained.Additionally, the volume of 400 myocytes was measured in each heart.Sections were stained with desmin and laminin antibodies and propidiumiodide (PI). Only longitudinally oriented cells with centrally locatednuclei were included. The length and diameter across the nucleus werecollected in each myocyte to compute cell volume, assuming a cylindricalshape (Olivetti et al., 1991; Beltrami et al., 1994). Myocytes weredivided in classes and the number of myocytes in each class wascalculated from the quotient of total myocyte class volume and averagecell volume (Kajstura et al., 1995; Reiss et al., 1996). Number ofarteriole and capillary profiles per unit area of myocardium wasmeasured as previously done (Olivetti et al., 1991; Beltrami et al.,1994).

Sections were incubated with BrdU or Ki67 antibody. Myocytes (M) wererecognized with a mouse monoclonal anti-cardiac myosin, endothelialcells (EC) with a rabbit polyclonal anti-factor VIII and smooth musclecells (SMC) with a mouse monoclonal anti-α-smooth muscle actin myosin.The fractions of M, EC and SMC nuclei labeled by BrdU and Ki67 wereobtained by confocal microscopy (Orlic et al., 2001). Nuclei sampled in11 cytokine-treated mice; BrdU: M=3,541; EC=2,604; SMC=1,824. Ki67:M=3,096; EC=2,465; SMC=1,404.

BrdU was injected daily between days 14 to 26 to measure the cumulativeextent of cell proliferation while Ki67 was assayed to determine thenumber of cycling cells at sacrifice. Ki67 identifies cells in G1, S,G2, prophase and metaphase, decreasing in anaphase and telophase (Orlicet al., 2001). The percentages of BrdU and Ki67 positive myocytes were1.6- and 1.4-fold higher than EC, and 2.8- and 2.2-fold higher than SMC,respectively (FIG. 18C, 19). The forming myocardium occupied 76±11% ofthe infarct; myocytes constituted 61±12%, new vessels 12±5% and othercomponents 3±2%. The band contained 15×10⁶ regenerating myocytes thatwere in an active growing phase and had a wide size distribution (FIGS.18D-E). EC and SMC growth resulted in the formation of 15±5 arteriolesand 348±82 capillaries per mm² of new myocardium. Thick wall arterioleswith several layers of SMC and luminal diameters of 10-30 μm representedvessels in early differentiation. At times, incomplete perfusion of thecoronary branches within the repairing myocardium during the fixationprocedure led to arterioles and capillaries containing erythrocytes(FIGS. 18F-H). These results provided evidence that the new vessels werefunctionally competent and connected with the coronary circulation.Therefore, tissue repair reduced infarct size and myocyte growthexceeded angiogenesis; muscle mass replacement was the prevailingfeature of the infarcted heart.

E. Determination of Cell Differentiation

Cytoplasmic and nuclear markers were used. Myocyte nuclei: rabbitpolyclonal Csx/NRx2.5, MEF2, and GATA4 antibodies (Orlic et al., 2001;Lin et al., 1997; Kasahara et al., 1998); cytoplasm: mouse monoclonalnestin (Kachinsky et al., 1995), rabbit polyclonal desmin (Hermann andAebi, 1998), cardiac myosin, mouse monoclonal α-sarcomeric actin andrabbit polyclonal connexin 43 antibodies (Orlic et al., 2001). ECcytoplasm: mouse monoclonal flk-1, VE-cadherin and factor. VIIIantibodies (Orlic et al., 2001; Yamaguchi et al., 1993; Breier et al.,1996). SMC cytoplasm: flk-1 and α-smooth muscle actin antibodies (Orlicet al., 2001; Couper et al., 1997). Scar was detected by a mixture ofcollagen type I and type III antibodies.

Five cytoplasmic proteins were identified to establish the state ofdifferentiation of myocytes (Orlic et al., 2001; Kachinsky et al., 1995;Hermann and Aebi, 1998): nestin, desmin, α-sarcomeric actin, cardiacmyosin and connexin 43. Nestin was recognized in individual cellsscattered across the forming band (FIG. 20A). With this exception, allother myocytes expressed desmin (FIG. 20B), α-sarcomeric actin, cardiacmyosin and connexin 43 (FIG. 20C). Three transcription factorsimplicated in the activation of the promoter of several cardiac musclestructural genes were examined (Orlic et al., 2001; Lin et al., 1997;Kasahara et al., 1998): Csx/NRx2.5, GATA-4 and MEF2 (FIGS. 21A-C).Single cells positive for flk-1 and VE-cadherin (Yamaguchi et al., 1993;Breier et al., 1996), two EC markers, were present in the repairingtissue (FIGS. 20D,E); flk-1 was detected in SMC isolated or within thearteriolar wall (FIG. 20F). This tyrosine kinase receptor promotesmigration of SMC during angiogenesis (Couper et al., 1997). Therefore,repair of the infarcted heart involved growth and differentiation of allcardiac cell populations resulting in de novo myocardium.

Example 3 Migration of Primitive Cardiac Cells in the Adult Mouse Heart

To determine whether a population of primitive cells was present in theadult ventricular myocardium and whether these cells possessed theability to migrate, three major growth factors were utilized aschemoattractants: hepatocyte growth factor (HGF), stem cell factor (SCF)and granulocyte monocyte colony stimulating factor (GM-CSF). SCF andGMCSF were selected because they have been shown to promotetranslocation of herriatopoietic stem cells. Although HGF inducesmigration of hematopoietic stem cells, this growth factor is largelyimplicated in mitosis, differentiation and migration of cardiac cellprecursors during early cardiogenesis. On this basis, enzymaticallydissociated cells from the mouse heart were separated according to theirsize. Methods for dissociating cardiac cells from heart tissue arewell-known to those skilled in the art and therefore would not involveundue experimentation (Cf U.S. Pat. No. 6,255,292 which is hereinincorporated by reference in its entirety) A homogenous population ofthe dissociated cardiac cells containing small undifferentiated cells,5-7 μm in diameter, with a high nucleus to cytoplasm ratio weresubjected to migration assay in Boyden microchambers characterized bygelatin-coated filters containing pores, 5 μm (Boyden et al., 1962, J.Exptl. Med. 115:453-456)

No major differences in the dose-response curve of migrated cells in thepresence of the three growth factors were detected. However, HGFappeared to mobilize a larger number of cells at a concentration of 100ng/ml. In addition, the cells that showed a chemotactic response to HGFconsisted of 15% of c-kit positive (c-kit^(POS)) cells, 50% of multidrugresistance-1 (MDR-1) labeled cells and 30% of stem cell antigen-1(Sca-1) expressing cells. When the mobilized cells were cultured in 15%fetal bovine serum, they differentiated into myocytes, endothelialcells, smooth muscle cells and fibroblasts. Cardiac myosin positivemyocytes constituted 50% of the preparation, while factor VIII labeledcells included 15%, alpha-smooth muscle actin stained cells 4%, andvimentin positive factor VIII negative fibroblasts 20%. The remainingcells were small undifferentiated and did not stain with these fourantibodies. In conclusion, the mouse heart possesses primitive cellswhich are mobilized by growth factors. HGF translocates cells that invitro differentiate into the four cardiac cell lineages.

Example 4 Cardiac C-Kit Positive Cells Proliferate In Vitro and GenerateNew Myocardium Vivo

To determine whether primitive c-kit^(POS) cells were present insenescent Fischer 344 rats, dissociated cardiac cells were exposed tomagnetic beads coated with c-kit receptor antibody (ACK-4-biotin,anti-c-kit mAb). Following separation, these small undifferentiatedcells were cultured in 10% fetal calf serum. Cells attached in a fewdays and began to proliferate at one week. Confluence was reached at7-10 days. Doubling time, established at passage P2 and P4, required 30and 40 hours, respectively. Cells grew up to P18 (90th generation)without reaching senescence. Replicative capacity was established byKi67 labeling: at P2, 88±14% of the cells contained Ki67 protein innuclei. Additional measurements were obtained between P1 and P4; 40% ofcells expressed alpha-sarcomeric actin or cardiac myosin, 13% desmin, 3%alpha-smooth muscle actin, 15% factor VIII1 or CD31, and 18% nestin.Under these in vitro conditions, cells showed no clear myofibrillarorganization with properly aligned sarcomeres and spontaneouscontraction was never observed. Similarly, Ang II, norepinephrine,isoprotererol, mechanical stretch and electrical field stimulationfailed to initiate contractile function. On this basis, it was decidedto evaluate whether these cells pertaining to the myogenic, smoothmuscle cell and endothelial cell lineages had lost permanently theirbiological properties or their role could be reestablished in vivo.Following BrdU labeling of cells at P2, infarcted Fischer 344 rats wereinjected with these BrdU positive cells in the damaged region, 3-5 hoursafter coronary artery occlusion. Two weeks later, animals weresacrificed and the characteristics of the infarcted area were examined.Myocytes containing parallel arranged myofibrils along theirlongitudinal axis were recognized, in combination with BrdU labeling ofnuclei. Moreover, vascular structures comprising arterioles andcapillary profiles were present and were also positive to BrdU. Inconclusion, primitive c-kit positive cells reside in the senescent heartand maintain the ability to proliferate and differentiate intoparenchymal cells and coronary vessels when implanted into injuredfunctionally depressed myocardium.

Example 5 Cardiac Stem Cells Mediate Myocyte Replication in the Youngand Senescent Rat Heart

The heart is not a post-mitotic organ but contains a subpopulation ofmyocytes that physiologically undergo cell division to replace dyingcells. Myocyte multiplication is enhanced during pathologic overloads toexpand the muscle mass and maintain cardiac performance. However, theorigin of these replicating myocytes remains to be identified.Therefore, primitive cells with characteristics of stem/progenitor cellswere searched for in the myocardium of Fischer 344 rats. Young and oldanimals were studied to determine whether aging had an impact on thesize population of stem cells and dividing myocytes. The numbers ofc-kit and MDR1 positive cells in rats at 4 months were 11±3, and18±6/100 mm² of tissue, respectively. Values in rats at 27 months were35±10, and 42±13/100 mm². A number of newly generated small myocyteswere identified that were still c-kit or MDR1 positive. Ki67 protein,which is expressed in nuclei of cycling cells was detected in 1.3±0.3%and 4.1±1.5% of myocytes at 4 and 27 months, respectively. BrdUlocalization following 6 or 56 injections included 1.0±0.4% and 4.4±1.2%at 4 months, and 4.0±1.5% and 16±4% at 27 months. The mitotic indexmeasured in tissue sections showed that the fraction of myocyte nucleiin mitosis comprised 82±28/10⁶ and 485±98/10⁶ at 4 and 27 months,respectively. These determinations were confirmed in dissociatedmyocytes to obtain a cellular mitotic index. By this approach, it waspossible to establish that all nuclei of multinucleated myocytes were inmitosis simultaneously. This information could not be obtained in tissuesections. The collected values showed that 95±31/ 10⁶ myocytes weredividing at 4 months and 620±98/10⁶ at 27 months. At both age intervals,the formation of the mitotic spindle, contractile ring, disassembly ofthe nuclear envelope, karyokinesis and cytokinesis were documented. Inconclusion, primitive undifferentiated cells reside in the adult heartand their increase with age is paralleled by an increase in the numberof myocytes entering the cell cycle and undergoing karyokinesis andcytokinesis. This relationship suggests that cardiac stem cells mayregulate the level and fate of myocyte growth in the aging heart.

Example 6 Chimerism of the Human Heart and the Role of Stem Cells

The critical role played by resident primitive cells in the remodelingof the injured heart is well appreciated when organ chimerism,associated with transplantation of a female heart in a male recipient,is considered. For this purpose, 8 female hearts implanted in male hostswere analyzed. Translocation of male cells to the grafted female heartwas identified by FISH for Y chromosome (see Example 1E). By thisapproach, the percentages of myocytes, coronary arterioles and capillaryprofiles labeled by Y chromosome were 9%, 14% and 7%, respectively.Concurrently, the numbers of undifferentiated c-kit and multidrugresistance-1 (MDR1) positive cells in the implanted female hearts weremeasured. Additionally, the possibility that these cells contained the Ychromosome was established. Cardiac transplantation involves thepreservation of portions of the atria of the recipient on which thedonor heart with part of its own atria is attached. This surgicalprocedure is critical for understanding whether the atria from the hostand donor contained undifferentiated cells that may contribute to thecomplex remodeling process of the implanted heart. Quantitatively, thevalues of c-kit and, MDR1 labeled cells were very low in controlnon-transplanted hearts: 3 c-kit and 5 MR1/100 mm² of left ventricularmyocardium. In contrast, the numbers of c-kit and MDR1 cells in theatria of the recipient were 15 and 42/100 mm² Corresponding values inthe atria of the donor were 15 and 52/100 mm² and in the ventricle 11and 21/100 mm². Transplantation was characterized by a marked increasein primitive undifferentiated cells in the heart. Stem cells in theatria of the host contained Y chromosome, while an average of 55% and63% of c-kit and MDR1 cells in the donor's atria and ventricle,respectively, expressed the Y chromosome. All c-kit and MDR1 positivecells were negative for CD45. These observations suggest that thetranslocation of male cells to the implanted heart has a major impact onthe restructuring of the donor myocardium. In conclusion, stem cells arewidely distributed in the adult heart and because of their plasticityand migration capacity generate myocytes, coronary arterioles andcapillary structures with high degree of differentiation.

Example 7 Identification and Localization of Stem Cells in the AdultMouse Heart

Turnover of myocytes occurs in the normal heart, and myocardial damageleads to activation of myocyte proliferation and vascular growth. Theseadaptations raise the possibility that multipotent primitive cells arepresent in the heart and are implicated in the physiological replacementof dying myocytes and in the cellular growth response following injury.On this basis, the presence of undifferentiated cells in the normalmouse heart was determined utilizing surface markers including c-kit,which is the receptor for stem cell factor, multidrug resistance-1(MDR1), which is a P-glycoprotein capable of extruding from the celldyes, toxic substances and drugs, and stem cell antigen-1 (Sca-1), whichis involved in cell signaling and cell adhesion. Four separate regionsconsisting of the left and right atria, and the base, mid-section andapical portion of the ventricle were analyzed. From the higher to thelower value, the number of c-kit positive cells was 26±11, 15±5, 10±7and 6±3/100 mm² in the atria, and apex, base and mid-section of theventricle, respectively. In comparison with the base and mid-section,the larger fraction of c-kit positive cells in the atria and apex wasstatistically significant. The number of MDR1 positive cells was higherthan those expressing c-kit, but followed a similar localizationpattern; 43±14, 29±16, 14±7 and 12±10/100 mm² in the atria, apex, baseand mid-section. Again, the values in the atria and apex were greaterthan in the other two areas. Sca-1 labeled cells showed the highestvalue; 150±36/100 mm² positive cells were found in the atria. Cellspositive for c-kit, MR1 and Sca-1 were negative for CD45, and formyocyte, endothelial cell, smooth muscle cell and fibroblast cytoplasmicproteins. Additionally, the number of cells positive to both c-kit andMDR1 was measured to recognize cells that possessed two stem cellmarkers. In the entire heart, 36% of c-kit labeled cells expressed MR1and 19% of MDR1 cells had also c-kit. In conclusion, stem cells aredistributed throughout the mouse heart, but tend to accumulate in theregions at low stress, such as the atria and the apex.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited by particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope thereof.

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1-160. (canceled)
 161. A method for restoring functional and structuralintegrity to damaged myocardium in a patient in need thereof comprising:(a) isolating adult hematopoietic stem cells from a bone marrowspecimen; and (b) administering an effective dose of said isolated adulthematopoietic stem cells to the border region of said damaged myocardiumin the patient's heart, wherein the functional and structural integrityof the damaged myocardium is restored following stem celladministration.
 162. The method of claim 161, wherein the isolated adulthematopoietic stem cells are lineage negative.
 163. The method of claim162, wherein the isolated adult hematopoietic stem cells are c-kitpositive.
 164. The method of claim 161, wherein the adult hematopoieticstem cells migrate into the damaged myocardium.
 165. The method of claim161, wherein the adult hematopoietic stem cells differentiate intomyocytes, endothelial cells, and smooth muscle cells.
 166. The method ofclaim 165, wherein the differentiated adult hematopoietic stem cellsproliferate.
 167. The method of claim 165, wherein the differentiatedadult hematopoietic stem cells assemble into myocardium and a coronaryartery.
 168. The method of claim 165, wherein the differentiated adulthematopoietic stem cells assemble into myocardium and an arteriole. 169.The method of claim 165, wherein the differentiated adult hematopoieticstem cells assemble into myocardium and a capillary.
 170. The method ofclaim 161, wherein the bone marrow specimen is obtained from the patientto which the isolated adult hematopoietic stem cells are administered.171. The method of claim 161, wherein the isolated adult hematopoieticstem cells are administered by injection.
 172. The method of claim 171,wherein the injection is intramyocardial.
 173. The method of claim 171,wherein the injection is trans-epicardial or trans-endocardial.
 174. Themethod of claim 161, wherein the isolated adult hematopoietic stem cellsare administered by a catheter.
 175. The method of claim 161, whereinthe effective dose of isolated adult hematopoietic stem cells is fromabout 2×10⁴ to 1×10⁵.
 176. The method of claim 161, further comprisingthe administration of an effective dose of one or more cytokines. 177.The method of claim 176, wherein the one or more cytokines is selectedfrom the group consisting of: (a) stem cell factor; (b)granulocyte-colony stimulating factor; (c) stromal cell-derivedfactor-1; (d) interleukin-3; (e) granulocyte-macrophage colonystimulating factor; (f) macrophage colony stimulating factor; (g)vascular endothelial cell growth factor; and combinations thereof. 178.The method of claim 177, wherein the administered cytokines are stemcell factor and granulocyte colony stimulating factor.
 179. The methodof claim 176, wherein the effective dose of one or more cytokines isfrom 50 μg/kg to 500 μg/kg.
 180. The method of claim 176, wherein theeffective dose of one or more cytokines is administered in multipleadministrations.
 181. The method of claim 180, wherein the multipleadministrations are given over the course of several days following theadministration of the isolated adult hematopoietic stem cells.
 182. Themethod of claim 176, wherein the effective dose of one or more cytokinesis administered systemically to the patient.
 183. The method of claim182, wherein the systemic administration is subcutaneous or intravenousinjection.
 184. The method of claim 176, wherein the administration ofone or more cytokines stimulates the patient's resident hematopoieticstem cells causing them to mobilize into the bloodstream.
 185. A methodfor restoring functional and structural integrity to damaged myocardiumin a patient in need thereof comprising: (a) isolating adulthematopoietic stem cells from a bone marrow specimen; (b) culturing saidisolated adult hematopoietic stem cells in vitro, wherein said adulthematopoietic stem cells proliferate and differentiate into myocytes,endothelial cells, and smooth muscle cells, thereby forming cardiacstructures; and (b) implanting said cardiac structures formed in vitrointo said damaged myocardium in the patient's heart, wherein theimplanted cardiac structures restore functional and structural integrityto the damaged myocardium.
 186. The method of claim 185, wherein thecardiac structures include myocardium, coronary arteries, arterioles,and/or capillaries.
 187. A method for restoring functional andstructural integrity to damaged myocardium in a patient in need thereofcomprising the administration of an effective dose of one or morecytokines selected from the group consisting of: a. a stem cell factor;b. a granulocyte-colony stimulating factor; c. a stromal cell-derivedfactor-1; d. an interleukin-3; e. a granulocyte-macrophage colonystimulating factor; f. a macrophage colony stimulating factor; g. avascular endothelial cell growth factor; and combinations thereof,wherein the functional and structural integrity of the damagedmyocardium is restored following cytokine administration.
 188. Themethod of claim 187, wherein the administered cytokines are stem cellfactor and granulocyte colony stimulating factor.